CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement CRISPR/Cas：一个获得诺贝尔奖的用于基因治疗和作物改良的精准基因组编辑技术 1 first-author https://orcid.org/0000-0002-0918-8156 2 3 corresp Hikmet BUDAK, hikmet.budak@icloud.com Hikmet BUDAK, hikmet.budak@icloud.com hikmet.budak@icloud.com https://orcid.org/0000-0002-2556-2478 4 corresp Baohong ZHANG, zhangb@ecu.edu Baohong ZHANG, zhangb@ecu.edu zhangb@ecu.edu https://orcid.org/0000-0002-9308-4340 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory for Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan430062, China Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory for Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Wuhan430062, China Agronomy Department, University of Florida, Gainesville, FL32611, USA Agronomy Department, University of Florida, Gainesville, FL32611, USA Montana BioAgriculture, Inc., Missoula, MT59802, USA Montana BioAgriculture, Inc., Missoula, MT59802, USA Department of Biology, East Carolina University, Greenville, NC27858, USA Department of Biology, East Carolina University, Greenville, NC27858, USA Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations. 自从CRISPR/Cas被确认为作为细菌先天性免疫的机制以来，CRISPR/Cas已迅速发展成为功能强大的多功能基因组编辑工具。其中包括开发为单碱基编辑器、引导编辑器、表观遗传编辑器、以及CRISPR干扰（CRISPRi）和CRISPR激活（CRISPRa）的基因调节工具。CRISPR/Cas也可以用于染色质和RNA靶向和成像。在许多生物学领域，尤其是在生物医学和农作物改良方面，CRISPR/Cas的应用已被广泛证明。作为一种新的疾病诊断工具，CRISPR可用于帮助检测和筛查人类和植物疾病，甚至在当前的新型冠状病毒型肺炎（COVID-19）大流行中也得到了应用。CRISPR/Cas也正在作为一种新的基因疗法以治疗包括癌症在内的各种人类疾病，并已帮助开发治疗某些疾病的药物。在农业育种方面，通过CRISPR/Cas精确靶向可以有效地调节关键的分子生物合成，从而用于修饰蛋白质、淀粉、油和其他功能成分来改善作物。除此之外，CRISPR/Cas也被广泛用于调控逆境胁迫相关的基因来显著增强植物对环境胁迫的耐受性和整体作物产量。提高CRISPR/Cas向细胞转化的效率和精度以及限制脱靶活性是该技术广泛应用的主要挑战。 Genome editing Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) Coronavirus disease 2019 (COVID-19) Cancer Precision breeding Crop improvement Gene knock-out/in Gene repair/replacement 基因编辑 CRISPR/Cas 新型冠状病毒型肺炎（COVID-19） 癌症 精准育种 作物改良 基因敲入和敲除 基因修复和替代 1 Introduction 1 Precision targeting of specific nucleotide sequences has been a long-standing dream in research and industrial fields, with potential applications in gene functional studies, gene therapies, and precision breeding of crops and domesticated animals. In the 1990s, discovery of genome editing meganucleases brought this dream into reality and initiated the beginning of a new era of targeted genome editing. Meganucleases are a class of endodeoxyribonucleases, which occur naturally in a variety of different organisms ( Silva et al., 2011 Silva et al., 2011 ). They function through the recognition and cleavage of specific double-stranded DNA (dsDNA) sequences, which are typically of > 14 bp in length, the sequence of which varies among different meganucleases. As the first class of molecular DNA "scissors" that were successfully used to edit genetic sequences precisely, meganucleases allowed previously unattainable targeted replacement, elimination, and modification of DNA. Also, the long length of the recognition site allowed meganucleases to have high target specificity and low off-target effects. However, this also created challenges, as there is a limited number of pre-defined targets and it is therefore hard to find a naturally occurring meganuclease that can target a chosen DNA sequence. Although scientists have attempted to modify meganucleases to broaden the editing site potential, construction is arduous and success has been limited. Several other nucleases have since been identified, and subsequently modified, for genome editing purposes. These include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). ZFNs and TALENs have similar properties, with both being composed of non-specific DNA cutting domains linked to specific DNA recognition domains. However, ZFNs recognize C2–H2 domains, whereas TALENs recognize DNA through transcription activator-like effectors (TALEs) ( Baker, 2012 Baker, 2012 ). Although both ZFNs and TALENs have been well used to target and edit specific genes, with some applications in clinical treatments ( Ellis et al., 2013 Ellis et al., 2013 ; Xu et al., 2014 Xu et al., 2014 ; Aravalli and Steer, 2016 Aravalli and Steer, 2016 ; Bañuls et al., 2020 Bañuls et al., 2020 ), they both have some shortcomings for genome editing. Like meganucleases, engineering ZFNs and TALENs to target desired sequences can be time-consuming, and often requires specialist knowledge ( Table 1 Table 1 ). ZFN: zinc finger nuclease; TALEN: transcription activator-like (TAL) effector nuclease; CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; dsDNA: double-stranded DNA; sgRNA: single guide RNA; PAM: protospacer-adjacent motif; DSB: double-stranded break. Genome editing tool Interaction Recognition site Required agents Required PAM InducingDSB Cell toxicity Specificity Meganucleases Protein‒DNA Large (12‒40 bp dsDNA) No Yes Low Very high ZFN Protein‒DNA Long ZFN with a Fok I DNA cleaving domain and a DNA-binding domain No Yes Low Very high TALEN Protein‒DNA Long TALEN with a Fok I DNA cleaving domain and a DNA-binding domain (TAL repeats) No Yes Low Very high CRISPR/Cas RNA‒DNA or RNA‒RNA Short Cas and sgRNA Yes Yes High High Genome editing tool Off-target Multiplex Editing efficiency Single nucleic acid targeting Cost 2 Easy for construction Meganucleases Low Difficult Low Yes High 2 No ZFN Low Difficult Relatively low Yes High 2 Relatively hard TALEN Low Difficult Relatively low Yes High 2 Relatively hard CRISPR/Cas Relatively high Yes High Yes Low 2 Simple, easy, and robust In comparison, the more recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system uses RNA-guided nucleases to create double-stranded breaks (DSBs) in DNA, and can be rapidly engineered to target almost any sequence with high efficiency, specificity, and ease of use ( Table 1 Table 1 ). Consequently, since its adaptation as a genome editing tool in 2012 ( Gasiunas et al., 2012 Gasiunas et al., 2012 ; Jinek et al., 2012 Jinek et al., 2012 ), CRISPR/Cas has quickly become the dominant genome and gene-editing tool for precision editing, with much research already underway in relation to its potentials for gene therapy and crop improvement. All of the above-mentioned methods have played significant roles in the history of precision genome editing, with Nature Methods (2012) listing meganucleases, ZFNs, and TALENs under the umbrella of molecular "scissors" as the Method of the Year in 2011, highlighting the significance of their development. All the methods are capable of knocking out/in individual genes, creating allelic mutations, changing gene regulatory controls, and adding reporters and epitope tags. Recently, the focus has shifted from their ability to edit genomes, to their efficiency and range. Each method has both benefits and drawbacks in its use. However, the perceived challenges to engineering meganucleases, ZFNs, and TALENs put them at a disadvantage, as it is not easy to design constructs to target the desired sequences. In this sense, the flexibility of CRISPR/Cas has allowed it to take the lead, and it has subsequently been used widely in applied and practical research. In recognition of this, CRISPR/Cas was selected by Science as the 2015 Breakthrough of the Year ( Travis, 2015 Travis, 2015 ), and in 2020, two pioneers of the CRISPR/Cas system, Dr. Emmanuelle CHARPENTIER and Dr. Jennifer A. DOUDNA, won the Nobel Prize in Chemistry for their work related to developing the technology. In this review, we focus on the CRISPR/Cas system in regard to its applications in the precision genome editing and crop improvement of a variety of plant species, particularly agriculturally important crops. Major problems associated with advancing the biotechnology will also be discussed alongside its future potential. 2 CRISPR/Cas classification and protospacer-adjacent motif requirements 1 The CRISPR/Cas system was first discovered in bacteria and archaea, where it functions as a form of adaptive immunity against viruses ( Ishino et al., 1987 Ishino et al., 1987 ; Nakata et al., 1989 Nakata et al., 1989 ; Hermans et al., 1991 Hermans et al., 1991 ; Mojica et al., 1993 Mojica et al., 1993 ; Jansen et al., 2002 Jansen et al., 2002 ; Mojica et al., 2005 Mojica et al., 2005 ). As an overview, Cas proteins typically recognize small motifs (about 3‒6 bp) present in the invading DNA, known as the protospacer-adjacent motif (PAM) ( Gleditzsch et al., 2019 Gleditzsch et al., 2019 ). The particular PAMs recognized differ among host species and are rarely present in the bacteria's own DNA to avoid self-cleavage ( Gleditzsch et al., 2019 Gleditzsch et al., 2019 ). Following PAM recognition, a segment of downstream DNA (about 20 bp), known as a protospacer, is copied out of the foreign DNA and into a CRISPR array for transcription into short CRISPR RNAs (crRNAs) ( Jinek et al., 2012 Jinek et al., 2012 ). These then anneal to trans-activating crRNAs (tracrRNAs) already present in the cell, which form a stem loop structure to allow Cas enzymes to bind ( Jinek et al., 2012 Jinek et al., 2012 ). The RNA complex then acts as a guide for the Cas endonuclease to initiate sequence specific cleavage of the foreign DNA, creating a DSB and silencing the pathogen ( Jinek et al., 2012 Jinek et al., 2012 ). When the system was adapted as a molecular tool, the crRNA/tracrRNA complex was replaced with a single guide RNA (sgRNA), which can be designed to target any desired sequence, and is introduced alongside a Cas protein to cleave the target site. Although the function of CRISPR/Cas has only recently been elucidated, scientists had been building up to the discovery for over 30 years. In 1987, whilst cloning an iap gene in Escherichia coli , Ishino et al. (1987) Ishino et al. (1987) accidently cloned a sequence which contained uncommon, interspaced repeats. Two years later, similarly repeated sequences were found in two bacteria closely related to E. coli , Shigella and Salmonella ( Nakata et al., 1989 Nakata et al., 1989 ), followed by the less closely related species, Mycobacterium tuberculosis , in 1991 ( Hermans et al., 1991 Hermans et al., 1991 ; Mojica et al., 1993 Mojica et al., 1993 ). Subsequently, scientists from research groups across the world began confirming that these sequences exist widely in both bacteria and archaea. In 2002, they were finally named as CRISPR ( Jansen et al., 2002 Jansen et al., 2002 ). However, CRISPRs were initially thought to function in genotyping ( Mojica and Montoliu, 2016 Mojica and Montoliu, 2016 ), and their true role was revealed only following the advancement of recombinant DNA technologies and the discovery of Cas proteins ( Mojica et al., 1995 Mojica et al., 1995 , 2005 2005 ; Jansen et al., 2002 Jansen et al., 2002 ). Upon detection of its DNA cleaving capabilities, CRISPR/Cas was quickly modified for use as a genome editing tool in 2012 ( Gasiunas et al., 2012 Gasiunas et al., 2012 ; Jinek et al., 2012 Jinek et al., 2012 ). Among different species of bacteria and archaea, a wide range of CRISPR/Cas systems have now been classified. These typically make use of different Cas endonucleases. Among these systems, Cas endonucleases show significant differences not only in their organization, but also in their size and functional structures. By using this diversity as a base for classification, it was recently suggested that CRISPR/Cas systems collectively form 2 classes, 6 types, and 33 subtypes ( Makarova et al., 2020 Makarova et al., 2020 ). Class I represents systems which generally contain multiple Cas enzymes collaboratively functioning to target DNA, and can be segregated into 3 types (I, III, and IV) and 16 subtypes (I-A, I-B, I-C, I-D, I-E, I-F, I-G, III-A, III-B, III-C, III-D, III-E, III-F, IV-A, IV-B, and IV-C). Due to the complexity of engineering and introducing multiple Cas enzymes into a cell, Class I systems are rarely used as genome editing tools. In comparison, Class II systems typically require only a single, large, multifunctional Cas enzyme, making them simpler for adaptation. As a result, much research has been expended on developing current Class II systems, and discovering more. Similar to Class I, in Class II there are currently 3 types (II, V, and IV) and 17 subtypes defined. Type II systems are the most well studied, following the early discovery of Cas9, which is currently the endonuclease most commonly used in CRISPR/Cas genome editing. However, research is ongoing, and the family is quickly expanding as more prokaryotes are explored. For example, in a recent study, Pausch et al. (2020) Pausch et al. (2020) discovered a new CRISPR/Cas system, termed CRISPR/CasΦ, in bacteriophages, which has a much smaller Cas endonuclease than previously observed (about 50% smaller than Cas9). In terms of genome editing, the smaller size may offer advantages, as it will be easier to deliver the molecule into cells. This highlights one way in which the inherent diversity is found in CRISPR/Cas, and allows the technology to be rapidly adapted for different purposes. Cas9 endonucleases found in Type II systems contain both an RuvC and an HNH nuclease domain, each of which is responsible for cleaving one strand of a dsDNA sequence, allowing a blunt-ended DSB. As Type II systems are so well-studied, they have been widely applied for a variety of purposes, including knock-in/out, base editing, transcriptional regulation, and gene imaging. Each CRISPR/Cas subtype contains many Cas enzymes with high similarity, typically obtained from different species. Although they show high evolutionary conservation, slight differences in the sequences can affect their activity, and overall size can greatly impact success. A good example of this is a comparison between SaCas9, derived from Staphylococcus aureus , and SpCas9, derived from Streptococcus pyogenes . SaCas9 has 1053 amino acid residues, making it much smaller than the more commonly used SpCas9 with 1368 amino acid residues. This difference makes SaCas9 more suitable for delivery in vivo ( Nishimasu et al., 2015 Nishimasu et al., 2015 ), and it has recently been shown to have high editing efficiency in mammalian and plant cells ( Ran et al., 2015 Ran et al., 2015 ; Steinert et al., 2015 Steinert et al., 2015 ; Kaya et al., 2016 Kaya et al., 2016 ; Xie HH et al., 2020 Xie HH et al., 2020 ). Differences in the sequence of Cas variants can also lead to distinct PAM preferences. SpCas9 recognizes a 5'-NGG-3' PAM. However, several other Cas9 orthologues have been characterized which have distinct recognition and targeting requirements. For example, NmCas9, derived from Neisseria meningitides , recognizes a 5'-N4GATT-3' PAM ( Esvelt et al., 2013 Esvelt et al., 2013 ; Hou et al., 2013 Hou et al., 2013 ), whereas St1Cas9 and St3Cas9, derived from Streptococcus thermophilus , both require either 5'-NNAGAAW-3' (where W represents A or T) or 5'-NGGNG-3' PAMs ( Garneau et al., 2010 Garneau et al., 2010 ; Magadán et al., 2012 Magadán et al., 2012 ; Müller et al., 2016 Müller et al., 2016 ). CjCas9, derived from Campylobacter jejuni , recognizes 5'-NNNNACAC-3' or 5'-NNNNRYAC-3' (R and Y represent purines (A/G) and pyrimidines (C/T), respectively) ( Kim E et al., 2017 Kim E et al., 2017 ), and ScCas9, derived from Streptococcus canis , prefers 5'-NNG-3' PAM ( Chatterjee et al., 2018 Chatterjee et al., 2018 ). CasX, another Type II derived from Deltaproteobacteria and Planctomycetes, has been shown to recognize 5'-TTCN-3' ( Burstein et al., 2017 Burstein et al., 2017 ). Lastly, SaCas9 recognizes a 5'-NNGRRT-3' PAM ( Nishimasu et al., 2015 Nishimasu et al., 2015 ; Ran et al., 2015 Ran et al., 2015 ). This provides great flexibility when identifying target sites for a desired edit, as different Cas variants provide greater scope. To further expand the scope of genome editing, several Cas9 nuclease variants have been engineered to introduce new PAM recognition sites. Cas9 VQR variant (D1135V/R1335Q/T1337R) recognizes 5'-NGA-3' PAM ( Kleinstiver et al., 2015b Kleinstiver et al., 2015b ; Hirano et al., 2016 Hirano et al., 2016 ; Hu XX et al., 2016 Hu XX et al., 2016 , 2018 2018 ). Cas9 EQR variant (D1135E/R1335Q/T1337R) recognizes 5'-NGAG-3' PAM ( Kleinstiver et al., 2015b Kleinstiver et al., 2015b ; Hirano et al., 2016 Hirano et al., 2016 ). Cas9 VRER variant (D1135V/G1218R/R1335E/T1337R) recognizes 5'-NGCG-3' PAM ( Kleinstiver et al., 2015b Kleinstiver et al., 2015b ; Hirano et al., 2016 Hirano et al., 2016 ). The xCas9 variant (A262T/R324L/S409I/E480K/E543D/M694I/E1219V) recognizes NG, GAA, and GTA PAMs ( Hu JH et al., 2018 Hu JH et al., 2018 ; Wang JJ et al., 2019 Wang JJ et al., 2019 ; Zhong et al., 2019 Zhong et al., 2019 ). SpCas9-NG variant (R1335V/L1111R/D1135V/G1218R/E1219F/A1322R/T1337R) recognizes 5'-NG-3' PAM ( Nishimasu et al., 2018 Nishimasu et al., 2018 ; Ge et al., 2019 Ge et al., 2019 ; Zhong et al., 2019 Zhong et al., 2019 ), and the SaCas9-KKH variant (E782K/N968K/R1015H) recognizes 5'-NNNRRT-3' PAM ( Kleinstiver et al., 2015a Kleinstiver et al., 2015a ). Two advances have recently been reported which further eliminate the constraint of PAM sequences in Cas9. Walton et al. (2020) Walton et al. (2020) developed two SpCas9 variants, named SpG and SpRY, that are able to recognize 5'-NGN-3' and 5'-NRN-3'/5'-NYN-3' (Y represents C or T) PAMs, respectively. Using phage-assisted non-continuous evolution, David LIU's group has also characterized three new SpCas9 variants, SpCas9 NRRH, SpCas9 NRCH, and SpCas9 NRTH, which collectively show preference for the 5'-NRNH-3' (H represents A, C, or T) PAM ( Miller et al., 2020 Miller et al., 2020 ). Class II, Type V CRISPR/Cas is the second most researched group and has proven effective in both animal and plant species. It is composed mostly of Cas12a (Cpf1) endonucleases ( Zetsche et al., 2015 Zetsche et al., 2015 ; Kim H et al., 2017 Kim H et al., 2017 ; Tang et al., 2017 Tang et al., 2017 ; Xu et al., 2017 Xu et al., 2017 ). These systems have been discovered in Acidaminococcus sp., Francisella tularensis subsp. novicida and Lachnospiraceae bacterium ( Zetsche et al., 2015 Zetsche et al., 2015 ; Tang et al., 2017 Tang et al., 2017 ). Although Cas12a is analogous to Cas9, it has unique features that differ from Cas9 and its orthologs. Firstly, cleavage via a Cas12a protein relies on a single RuvC-like endonuclease domain, not the RuvC and HNH combination found in Type II ( Zetsche et al., 2015 Zetsche et al., 2015 ). Secondly, Cas12a cleaves target DNA using only crRNAs, rather than a crRNA/tracrRNA complex ( Zetsche et al., 2015 Zetsche et al., 2015 ). Thirdly, CRISPR/Cas12a genome editing depends on recognition of a T-rich PAM sequence (5'-TTTV-3' PAM) ( Zetsche et al., 2015 Zetsche et al., 2015 ). Together, this creates a 4- or 5-nt 5'-staggered DSB at targeted sites, which facilitates more precise gene replacement mediated by the non-homologous end joining (NHEJ) DNA repair pathway ( Zetsche et al., 2015 Zetsche et al., 2015 ). As with Cas9, several Cas12a variants have been identified for engineered genome editing. The AsCas12a RR variant (S542R/K607R) and AsCas12a RVR variant (S542R/K548V/N552R) recognize 5'-TYCV-3' and 5'-TATV-3' PAMs, respectively ( Gao et al., 2017 Gao et al., 2017 ). In comparison, the LbCas12a RR variant (G532R/K595R) recognizes 5'-CCCC-3' and 5'-TYCV-3' PAMs and the LbCas12a RVR variant (G532R/K538V/Y542R) recognizes 5'-TATV-3' PAM ( Li SY et al., 2018 Li SY et al., 2018 ; Zhong et al., 2018 Zhong et al., 2018 ). Among the Class II systems, Type VI is the most distinct. Cas13 dominates Type VI and contains neither an RuvC nor an HNH nuclease domain. Instead, Cas13 contains two HEPN domains which target RNA instead of DNA, opening up a novel avenue for epigenetic editing. In addition, although PAM recognition is a requirement for most CRISPR/Cas systems, the ability to target RNA mitigates this requirement in proteins such as Cas13. However, to date, Cas13 is the only Cas found that can directly target RNA sequences ( Makarova et al., 2020 Makarova et al., 2020 ). 3 CRISPR/Cas precision genome editing 1 3.1 Repair pathways 2 Once DSBs have been formed by CRISPR/Cas in DNA, cells typically undergo one of two main repair mechanisms. The most commonly used pathway is NHEJ, in which a DNA ligase links the two broken DNA ends together to reconnect the sequence. During this process, it is common for one or more nucleotides to be inserted and/or deleted at the DSB site. Due to this, NHEJ often results in either a frameshift in the DNA that changes the amino acid sequence or a nonsense mutation. Thus, CRISPR/Cas is efficient at inducing gene mutations and is widely used for functional studies of individual genes. DNA can also be repaired via homology-directed repair (HDR). HDR is an error-free repair pathway in which a homologous DNA template, typically obtained from a gene copy or homologous gene, is inserted to fill the DSB and reform the original DNA sequence. As understanding of these repair mechanisms has increased, researchers have been able to add artificial DNA templates into cells, such as whole genes with arms homologous to the DSB ( Heyer et al., 2010 Heyer et al., 2010 ), allowing the insertion of specific DNA sequences. This facilitates overexpression studies, as it allows targeted gene insertion at precise DNA locations, a feat which cannot be achieved with traditional transgene technologies. Although both NHEJ and HDR can be used to repair DSBs generated by CRISPR/Cas, NHEJ is the most common ( Feng et al., 2021 Feng et al., 2021 ). The HDR pathway is used much less often because of its lower frequency and efficiency, even when a donor DNA template is present. Many factors can affect this efficiency, including cell type, cell cycle stage, concentration of the donor DNA template ( Dickinson et al., 2013 Dickinson et al., 2013 ), length of the homologous arms, the CRISPR/Cas system (as discussed above), and the delivery system ( Lin et al., 2014 Lin et al., 2014 ). HDR efficiency was dramatically increased in HEK293T human primary neonatal fibroblast and human embryonic stem cells relative to experiments with unsynchronized cells, with rates of HDR of up to 38% observed ( Lin et al., 2014 Lin et al., 2014 ), and dividing rather than nondividing cells. In addition, controlled cell cycling provided a 6-fold increase in the ratio of HDR to NHEJ repair pathways in quiescent stem cells ex vivo and in vivo ( Shin et al., 2020 Shin et al., 2020 ). Co-transfection of a CRISPR/Cas system with RAD51, a key molecule during the initial step of HDR, also increased HDR efficiency 2.5-fold in layers 2/3 of pyramidal neurons in the somatosensory cortex of mouse brains ( Kurihara et al., 2020 Kurihara et al., 2020 ). Inhibition of DNA ligase IV (LIG4), an essential molecule for NHEJ, increased efficiency of HDR-mediated knock-in ( Cao et al., 2020 Cao et al., 2020 ). Overall, inhibition of LIG4 appeared more effective than RAD51 overexpression for induced HDR-mediated knock-in ( Cao et al., 2020 Cao et al., 2020 ). HDR-mediated genome editing has been reported in both plants and animals. Generally, HDR-mediated genome editing in plants has been achieved with only up to 10% success, although there are few reports of high efficiency ( Li, 2009 Li, 2009 ; Li et al., 2013 Li et al., 2013 ; Sauer et al., 2016 Sauer et al., 2016 ). Efficiency of 30% or even higher can be achieved after certain modifications, such as using single-stranded oligodeoxynucleotides (ssODNs) instead of a plasmid donor in animals ( Yang et al., 2014 Yang et al., 2014 ). Thus, it is much easier to obtain HDR-mediated genome editing in animal cells than in plant cells. One route which is being explored to increase efficiency and specificity is modifying the Cas endonucleases. To date, much of this research has been focused on modifying the cutting domains in Cas9. Loss function of a single cutting domain, known as nick Cas (nCas), or both domains, known as dead Cas (dCas), has been shown not to affect Cas-binding activity. Also, loss of function of one domain does not affect the cutting activity of the other. These modified Cas proteins can then be fused with other functional proteins, which has paved the way towards new CRISPR/Cas genome editors with expanded purposes, including recently developed base editors and prime editors ( Table 2 Table 2 ). CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; CRISPRi: CRISPR interference; CRISPRa: CRISPR activator; dCas: dead Cas; nCas: nick Cas; DSB: double-stranded break; SSB: single-stranded break; gRNA: guide RNA; sgRNA: single guide RNA; pegRNA: prime editing guide RNA. CRISPR/Cas genome/gene editors Cas enzyme Additional enzyme Induced DSB or SSB Require DNA template gRNA Major application Regular CRISPR/Cas editor without DNA template Cas DSB No sgRNA Gene knock-out, inducing silence mutations Regular CRISPR/Cas editor with DNA template Cas DSB Yes sgRNA Gene knock-in, DNA replacement CRISPR/Cas epigenetic editor dCas Epigenetic modifier, including DNA methyltransferase No No sgRNA Regulation of epigenome and gene expression CRISPRi editor dCas dCas alone or fusing with a repressor No No sgRNA Knock-down CRISPRa editor dCas Fusing an effector with dCas9 No No sgRNA Activation of gene expression Base editor dCas Nucleobase deaminase enzyme No No sgRNA Base change Prime editor nCas Reverse transcriptase SSB No pegRNA Sequence repair 3.2 Base editors 2 Base editors are capable of creating specific base changes. This is achieved by fusing a dCas9 nuclease with an engineered base converter enzyme, which mediates the conversion of one base to another under the guidance of a sgRNA. Currently, three kinds of base editors have been generated. The first, cytosine base editors (CBEs), catalyze the conversion of a single C to a T, which in turn initiates a change from a C/G pair to a T/A pair. This is accomplished by fusing dCas9 with cytidine deaminase and uracil glycosylase inhibitor (UGI) domains ( Komor et al., 2016 Komor et al., 2016 ; Li XS et al., 2018 Li XS et al., 2018 ; Zong et al., 2018 Zong et al., 2018 ). The next category is adenine base editors (ABEs), which consists of dCas9 fused with an evolved transfer RNA adenosine deaminase (TadA * ). This converts an A to a G, initiating an A/T to G/C base pair switch ( Gaudelli et al., 2017 Gaudelli et al., 2017 ; Kang et al., 2018 Kang et al., 2018 ; Hua et al., 2020 Hua et al., 2020 ). Lastly, and most recently, two independent research groups have established two new base editors, known as glycosylase base editors (GBEs) and C-to-G base editors (CGBEs), which convert C-to-A and C-to-G, respectively ( Kurt et al., 2020 Kurt et al., 2020 ; Zhao et al., 2020 Zhao et al., 2020 ). Base editor tools are rapidly being developed to achieve even higher base conversion efficiencies and broader activity window spans. To date, there have been four generations of CBE base editors developed, each with optimized sequence composition and cytidine deaminases or derivatives, including BE1, BE2, BE3, BE3-YE1/BE3-YE2/BE3-YEE/BE3-EE, and BE4 ( Komor et al., 2016 Komor et al., 2016 , 2017 2017 ; Kim YB et al., 2017 Kim YB et al., 2017 ; Gehrke et al., 2018 Gehrke et al., 2018 ; Endo et al., 2019 Endo et al., 2019 ; Wu Y et al., 2019 Wu Y et al., 2019 ; Doman et al., 2020 Doman et al., 2020 ; Tan JJ et al., 2020 Tan JJ et al., 2020 ). In addition, for ABE base editors, ABE7.10, ABE8, and ABE8e have been reported to have high catalytic activity for installing A/T to G/C point mutations ( Gaudelli et al., 2017 Gaudelli et al., 2017 , 2020 2020 ; Ren et al., 2019 Ren et al., 2019 ; Li C et al., 2020 Li C et al., 2020 ; Richter et al., 2020 Richter et al., 2020 ). Although base editors have been developed to accomplish conversion of C/G to T/A, A/T to G/C, C-to-A, and C-to-G, they are still constrained by their ability to insert, delete, or create other transversion mutations, such as G to C and G to T ( Komor et al., 2016 Komor et al., 2016 ; Nishida et al., 2016 Nishida et al., 2016 ; Gaudelli et al., 2017 Gaudelli et al., 2017 ; Kim YB et al., 2017 Kim YB et al., 2017 ; Anzalone et al., 2019 Anzalone et al., 2019 ; Chen et al., 2019 Chen et al., 2019 ; Lin et al., 2020 Lin et al., 2020 ). 3.3 Prime editors 2 In 2019, David LIU's group at Harvard University developed a new CRISPR/Cas genome editing system known as a prime editor, by fusing a reverse transcriptase (RT) to a catalytically impaired Cas9 endonuclease (CRISPR/dCas9 H840A) ( Anzalone et al., 2019 Anzalone et al., 2019 ). Prime editing is a search-and-replace genome editing technology that directly inputs a new genome sequence without a DNA template in the targeted genome site, and is revolutionizing this field. Prime editing systems harbor a programmable prime editing guide RNA (pegRNA) that functions to produce sgRNAs and RT templates of desired edits ( Anzalone et al., 2019 Anzalone et al., 2019 ). These systems have been demonstrated to work in both human and plant cells, including wheat and rice ( Anzalone et al., 2019 Anzalone et al., 2019 ; Li HY et al., 2020 Li HY et al., 2020 ; Lin et al., 2020 Lin et al., 2020 ; Tang et al., 2020 Tang et al., 2020 ; Xu et al., 2020 Xu et al., 2020 ). Compared to classic CRISPR/Cas systems, prime editing systems have the capability to accomplish targeted DNA insertions, deletions, and 12 types of base conversions, without generating DSBs or needing donor DNA templates ( Anzalone et al., 2019 Anzalone et al., 2019 ). These hold great potential for the future of genome editing, but the editing efficiency of prime editors is closely determined by the design of an appropriate pegRNA, and so requires specific knowledge to obtain success ( Anzalone et al., 2019 Anzalone et al., 2019 ). Prime editors are a new type of genome editor, many details still need to be modified, and more studies are required to realize their full potential. This new technology was first developed in animal models and can achieve a reasonable genome editing efficiency of up to 50% in human cells ( Anzalone et al., 2019 Anzalone et al., 2019 ). Although it has been used successfully to edit plant genes, the editing efficiency is much lower in plants than in animals ( Anzalone et al., 2019 Anzalone et al., 2019 ; Li HY et al., 2020 Li HY et al., 2020 ; Lin et al., 2020 Lin et al., 2020 ; Tang et al., 2020 Tang et al., 2020 ; Xu et al., 2020 Xu et al., 2020 ). Also, more targeted genes should be tested in both plants and animals in the future. 3.4 Epigenome editors 2 As understanding of the factors regulating genetic processes has improved, epigenetics has been revealed as an extremely important factor for controlling biological pathways. This has led to it becoming a novel target for clinical therapy and crop improvement. DNA methylation, histone modification, and non-coding RNAs are all primary epigenetic factors that aid in controlling gene expression. The extent of their control is being explored via CRISPR/Cas editing. It is well known that DNA methylation is a major epigenetic modifier in plants and animals (Bogdanović and Lister, 2017). Methylation plays a role in controlling many biological and metabolic processes, including cell identity (Bogdanović and Lister, 2017), tissue differentiation and development ( Zhang HM et al., 2018 Zhang HM et al., 2018 ), response to environmental stresses ( Zhang HM et al., 2018 Zhang HM et al., 2018 ), fruit ripening ( Huang et al., 2019 Huang et al., 2019 ), and cell wall biosynthesis and formation ( Haas et al., 2020 Haas et al., 2020 ; Zhang and Zhang, 2020a Zhang and Zhang, 2020a ). The term DNA methylation encompasses the process whereby specific DNA sequences become methylated or demethylated, either inhibiting or promoting gene expression, respectively. With this in mind, regulating DNA methylation represents a novel pathway for controlling gene expression and subsequent gene function. DNA methylation is initiated by DNA methyltransferase (DNMT), so controlling DNMT activity should in turn control DNA methylation. By fusing dCas9 to a catalytic domain of DNMT, such as DNMT3 or MQ3, CRISPR/Cas systems have successfully targeted methylation to specific sites in both plants and animals ( Liu et al., 2016 Liu et al., 2016 ; Vojta et al., 2016 Vojta et al., 2016 ; Lau and Suh, 2018 Lau and Suh, 2018 ). By fusing dCas to a demethylation enzyme, such as a ten-eleven translocation (TET) family member, the CRISPR/dCas system may also be used to remove methyl groups from specific DNA sequences, initiating increased expression of the target gene. Lau and Suh (2018) Lau and Suh (2018) successfully demonstrated this by targeting a CGG expansion mutation in the 5'-untranslated region (5'-UTR) of the fragile X mental retardation 1 ( FMR1 ) gene in Fragile X syndrome (FXS) patients, which is the most common form of genetic intellectual disability in males. Several studies have shown CRISPR/dCas9-TET1 capable of removing up to 90% of targeted methylations, significantly increasing gene expression at the target sites ( Liu et al., 2016 Liu et al., 2016 ; Morita et al., 2016 Morita et al., 2016 ; Hanzawa et al., 2020 Hanzawa et al., 2020 ; Horii et al., 2020 Horii et al., 2020 ). Horii et al. (2020) Horii et al. (2020) also used this technology successfully to generate an epigenetic disease model in mice via targeted demethylation of the epigenome. DNA in cells is wrapped in chromatin fibers which are held together with histone proteins that keep the DNA condensed. Thus, histones play an important role in controlling gene expression. Many studies have shown that both histone methylation and acetylation affect chromatin and histone remodeling, which in turn affects gene expression ( Strahl and Allis, 2000 Strahl and Allis, 2000 ; Adli, 2018 Adli, 2018 ). Several of these studies have focused on the enzymes associated with histone methylation and acetylation, to further elucidate gene regulation. One such enzyme, lysine-specific demethylase 1 (LSD1), is a histone demethylase that selectively removes one or two methyl groups from histone H3 at the Lys4 position ( Hayward and Cole, 2016 Hayward and Cole, 2016 ). By fusing dCas9 with LSD1, Kearns et al. (2015) Kearns et al. (2015) were able to successfully target active enhancer markers, H3K4me2 and H3K27ac, and alter the expression profiles of target genes ( Kearns et al., 2015 Kearns et al., 2015 ). In addition, by fusing a dCas9 with a transcriptional repressor histone deacetylase 1 (HDAC1), Liu et al. (2020) Liu et al. (2020) successfully silenced the KRAS gene, one of the most frequently mutated oncogenes in cancer patients. With the KRAS mutation, cell growth was significantly inhibited and the cell death was increased in cancer cells ( Liu et al., 2021 Liu et al., 2021 ). Non-coding RNAs have recently been revealed as important epigenetic gene regulators. In particular, small non-coding RNAs such as microRNAs (miRNAs), have been highlighted to play diverse roles in development and stress responses in plants and animals ( Zhang et al., 2007b Zhang et al., 2007b ; Gebert and MacRae, 2019 Gebert and MacRae, 2019 ), including serving as oncogenes and tumor suppressors ( Zhang et al., 2007a Zhang et al., 2007a ). Due to their new-found significance, a lot of attention has been placed on developing methods to edit individual miRNA genes, as the function of many miRNAs is still unclear. Using CRISPR/Cas9, Huo et al. (2017) Huo et al. (2017) were able to successfully knock out the miRNA-21 (miR21) gene in ovarian cancer SKOV3 and OVCAR3 cell lines. The edited cell lines revealed that the miR21 knock-out significantly inhibited cancer cell proliferation, migration, and invasion, highlighting a prime target for cancer gene therapies ( Huo et al., 2017 Huo et al., 2017 ). To add to this, again using CRISPR/Cas9, Jiang et al. (2020) Jiang et al. (2020) were able to knock out several miRNA genes, including miR30c, miR205, and miR663a, in human LNCaP cell lines, and were able to determine that miR-1225-5p and miR-663a knock-outs decreased lactate production in LNCaP cells. However, although miRNA-related research is a quickly developing field with thousands of miRNAs already identified, most research has been focused on humans, and there is still much to learn. With the application of CRISPR/Cas genome editing, information can be obtained in a specific, targeted manner, accelerating progress towards miRNAs as targets for clinical treatments and crop improvement. 3.5 CRISPRi and CRISPRa editors 2 There are many factors that affect gene expression, including binding of transcription factors and associated proteins to promotor regions during transcription/translation. By targeting Cas enzymes to such regions, either alone or with fused elements such as trans-effectors, gene expression may be repressed or enhanced. This represents a new role for CRISPR/Cas, termed CRISPRa for enhancing gene expression and CRISPRi for inhibiting gene expression. An early study showed that catalytically dCas9 alone could strongly bind to target DNA and interfere with the binding activity of other proteins, such as RNA polymerase and transcription factors, blocking transcription initiation and elongation in E. coli and mammalian cells ( Qi et al., 2013 Qi et al., 2013 ). The efficiency of CRISPRi strongly depended on sgRNA quality and target site efficiency, although it was shown that targeting any part of a gene, including the promoter, transcript sequence, and even UTR regions, could provide up to 86% repression with dCas9 alone ( Lawhorn et al., 2014 Lawhorn et al., 2014 ). The most active sgRNAs used were shown to repress gene expression by up to 99% ( Gilbert et al., 2014 Gilbert et al., 2014 ). Thus, screening and modifying sgRNAs is very important for achieving high repression efficiency using CRISPRi technology. CRISPRi-based inhibition can be significantly enhanced by fusing dCas9 with a gene expression repressor, such as the Krüppel-associated box (KRAB) domain of Kox1, the chromoshadow (CS) domain of HP1α, the WPRW domain of Hes1, or four concatenated copies of the mSin3 interaction domain (SID4X), in a variety cells, including human cells ( Gilbert et al., 2013 Gilbert et al., 2013 ; Konermann et al., 2013 Konermann et al., 2013 ). Among all tested gene expression repressors, dCas9-KRAB was the most effective at repressing expression of a target gene ( Gilbert et al., 2013 Gilbert et al., 2013 ; Konermann et al., 2013 Konermann et al., 2013 ). In comparison, CRISPRa is usually composed of dCas9 fused with a transcriptional activator, allowing targeted enhancement of gene expression. This was demonstrated by Maeder et al. (2013) Maeder et al. (2013) , who fused a dCas9 with a transcriptional activation domain from the herpes virus activator VP64 to successfully increase gene expression levels in human cells. This technology has also been used to activate multiple endogenous genes ( IL1RN , SOX2 , and OCT4 ) in both human and mouse cells. To date, the highest efficiency for CRISPRa activation was obtained by using multiple sgRNAs, which suggests a synergistic relationship during gene regulation ( Cheng et al., 2013 Cheng et al., 2013 ). CRISPRa-targeting sites may also affect CRISPRa efficiency. Cheng et al. (2013) Cheng et al. (2013) demonstrated that CRISPRa binding to the proximal region of a promoter had a particularly high activation efficiency for the target gene. Binding to tandem arrays, such as SunTag, can also significantly enhance CRISPRa-based gene expression through recruiting multiple activator copies ( Tanenbaum et al., 2014 Tanenbaum et al., 2014 ). Fusing multiple gene expression activators with dCas9 also increased CRISPRa activity and significantly enhanced gene expression ( Chavez et al., 2015 Chavez et al., 2015 ; Konermann et al., 2015 Konermann et al., 2015 ). In addition, Joung et al. (2017) Joung et al. (2017) were able to use the technology to screen and identify a long noncoding RNA (lncRNA) locus that regulates a gene neighborhood, highlighting a novel use for CRISPRa in identifying epigenetic relationships. Both CRISPRi and CRISPRa are highly specific and can be used to repress or activate multiple target genes simultaneously. In addition, the effects are reversible and have no detectable off-target effects ( Gilbert et al., 2013 Gilbert et al., 2013 ; Qi et al., 2013 Qi et al., 2013 ). CRISPR/Cas technology has also been adapted for use in live cell chromatin imaging ( Ma et al., 2015 Ma et al., 2015 ; Fu et al., 2016 Fu et al., 2016 ; Wu XT et al., 2019 Wu XT et al., 2019 ; Khosravi et al., 2020 Khosravi et al., 2020 ) and manipulation of chromatin topology ( Adli, 2018 Adli, 2018 ; Yim et al., 2020 Yim et al., 2020 ). 4 Wide use of CRISPR/Cas in clinical and preclinical research as well as disease treatment 1 Since it was considered a genome editing tool, CRISPR/Cas has attracted huge attention from scientific communities and industries for treating human genetic diseases. In the past ten years, great progress has been achieved in using CRISPR/Cas genome editing technology clinically and preclinically to treat human genetic disorders and screen and diagnose human diseases, and for fundamental studies in biomedicine. 4.1 Using CRISPR/Cas to screen and detect diseases 2 Early screening and detection of different diseases, particularly deadly diseases, is very important for disease treatment. In recent decades, many different technologies have been developed to screen, detect, and diagnose various diseases, including cancers. Due to its unique characteristics, CRISPR has been quickly developed and adopted as a diagnostic tool for different human diseases. Many human diseases are genetic disorders or are associated with a specific gene mutation in the human genome. All bacterial and viral diseases, such as the current coronavirus disease 2019 (COVID-19), are associated with specific nucleic acid sequences from the pathogen. These sequences provide perfect targets for the CRISPR/Cas system to produce a readable signal, termed a biomarker, for screening, detecting, and diagnosing various diseases. COVID-19 is currently the most infectious disease, and also is one of the deadliest infectious diseases in human history ( Shi et al., 2020 Shi et al., 2020 ). It is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By December 2020, SARS-CoV-2 had infected more than 70 million people and was responsible for more than 1.5 million deaths. Currently, the most reliable method for diagnosing COVID-19 is by using polymerase chain reaction (PCR) to detect SARS-CoV-2 sequences. However, CRISPR/Cas-based diagnostic tools may have advantages for detecting different pathogens, including the SARS-CoV-2 virus, due to their higher sensitivity and specificity compared to traditional PCR and real-time quantitative reverse transcription-PCR (qRT-PCR) ( Kumar et al., 2020 Kumar et al., 2020 ). CRISPR/Cas-based technology can be used to distinguish a single nucleotide difference and thus has high specificity for detecting different genetic variations, even with a single nucleotide polymorphism (SNP). CRISPR/Cas-based disease diagnostics may reshape the profiles of global diagnostic and health care systems ( Gootenberg et al., 2017 Gootenberg et al., 2017 ; Chertow, 2018 Chertow, 2018 ; Kumar et al., 2020 Kumar et al., 2020 ). Recently, the CRISPR/Cas12a system was also developed as a CRISPR/Cas-mediated lateral flow nucleic acid assay (CASLFA) for rapidly and sensitively detecting different pathogens, including COVID-19 ( Wang XJ et al., 2020b Wang XJ et al., 2020b ). A CRISPR/Cas12a fluorescent cleavage assay coupled with recombinase polymerase amplification was also developed for sensitive and specific detection of pathogens ( Kanitchinda et al., 2020 Kanitchinda et al., 2020 ). Other CRISPR/Cas-based diagnostic tools, such as SHERLOCH (specific high-sensitivity enzymatic reporter unlocking) and DETECTR (DNA endonuclease-targeted CRISPR trans reporter), have also been developed for detecting emerging infectious diseases, including COVID-19 ( Gootenberg et al., 2017 Gootenberg et al., 2017 ; Chen et al., 2018 Chen et al., 2018 ; Kellner et al., 2019 Kellner et al., 2019 ; Li ZJ et al., 2020 Li ZJ et al., 2020 ; Brandsma et al., 2021 Brandsma et al., 2021 ; Mustafa and Makhawi, 2021 Mustafa and Makhawi, 2021 ). This novel technology can detect as few as ten copies of virus genes in less than one hour. The principle of CRISPR/Cas-based diagnostics is based on collateral cleavage activity, in which Cas12a/Cas13 nucleases become activated after the crRNA-targeted cleavage. After they are activated, Cas cleaves the nearby single-stranded DNA (ssDNA)/RNA molecules non-specifically, a feature called collateral cleavage or trans-cleavage ( Kumar et al., 2020 Kumar et al., 2020 ; Wang M et al., 2020 Wang M et al., 2020 ). There have been several reports on diagnostics and detection of SARS-CoV-2 using CRISPR/Cas-based therapeutic tools ( Chen et al., 2018 Chen et al., 2018 ; Ali et al., 2020 Ali et al., 2020 ; Broughton et al., 2020 Broughton et al., 2020 ; Ding et al., 2020 Ding et al., 2020 ; Islam and Iqbal, 2020 Islam and Iqbal, 2020 ; Javalkote et al., 2020 Javalkote et al., 2020 ; Wang M et al., 2020 Wang M et al., 2020 ; Wang XJ et al., 2020b Wang XJ et al., 2020b ). Apart from COVID-19, CRISPR/Cas-based therapeutic tools have also been used to detect and diagnose other human disease-causing pathogens, including the Zika, West Nile, and yellow fever viruses ( Gootenberg et al., 2018 Gootenberg et al., 2018 ; Myhrvold et al., 2018 Myhrvold et al., 2018 ; Quan et al., 2019 Quan et al., 2019 ), human papillomavirus (HPV), M. tuberculosis , and Salmonella ( Ai et al., 2019 Ai et al., 2019 ; Wang XJ et al., 2020a Wang XJ et al., 2020a ). CRISPR/Cas-based therapeutic tools can also be used to diagnose non-infectious diseases, including cancers and genetic disorders ( Tian et al., 2019 Tian et al., 2019 ). 4.2 Using CRISPR/Cas to study pathogenesis and the related mechanisms as well as disease treatment 2 Many human diseases are associated with genetic information. Some are associated with endogenous genetic disorders, whereas others are associated with exogenous genetic infection, such as virus-infected diseases. Because CRISPR/Cas specifically targets a DNA/RNA sequence it can be used to monitor, break down, replace, or regulate target sequences for various purposes. CRISPR/Cas technology has attracted huge attention from both academic communities and biotechnological industries to treat various human diseases since it was recognized as a natural genome editing tool in 2012. Currently, CRISPR/Cas has huge potential applications for studying and treating human diseases, particularly genetic disorders. 4.2.1　Creating animal genetic models for treating human genetic diseases 3 A good animal model will allow us to better understand targeted human diseases not only in relation to their pathogenesis and mechanisms, but also for screening drugs and studying their potential side effects. Since it can precisely target an individual gene for editing with high efficiency and fewer side effects, CRISPR/Cas genome editing, including base editing and prime editing, has been quickly adopted to generate a range of animal models for studying various human genetic diseases. In a recent literature survey, Zhang (2021) Zhang (2021) found that at least a dozen animal models have been generated using CRISPR/Cas genome editing technology. These include mouse, rat, pig, and rabbit models for studying various human diseases, including human non‐small‐cell lung cancers (NSCLCs) ( Maddalo et al., 2014 Maddalo et al., 2014 ), hepatocellular carcinoma (HCC) ( Liu et al., 2017 Liu et al., 2017 ), Hutchinson-Gilford progeria syndrome (HGPS) ( Liu ZQ et al., 2018 Liu ZQ et al., 2018 ), corneal dystrophy ( Kitamoto et al., 2020 Kitamoto et al., 2020 ), and X-linked dilated cardiomyopathy (XLCM) ( Liu ZQ et al., 2018 Liu ZQ et al., 2018 ). Fujihara et al. (2020) Fujihara et al. (2020) successfully created a rat model by using CRISPR/Cas9 genome editing for studying complex behavioral changes during schizophrenia. Schizophrenia is a serious mental disease that affects how a person thinks, feels, and behaves, and causes lots of problems for patients and their families. However, no effective treatments are available for schizophrenia. Genetic evidence has shown that GABAergic dysfunction is associated with the pathogenesis of schizophrenia, and the γ-aminobutyric acid(GABA) synthetic enzyme glutamate decarboxylase 67-kDa isoform (GAD67) is downregulated in the brains of patients with the disease ( Fujihara et al., 2020 Fujihara et al., 2020 ). GAD67 is encoded by the GAD1 gene. Some SNPs surrounding the GAD1 gene were found to be associated with schizophrenia in North America and China ( Addington et al., 2005 Addington et al., 2005 ; Du et al., 2008 Du et al., 2008 ). Rats with a CRISPR knock-out of Gad1 produced about 48% less GABA than wild-type rats, and showed a wide range of behavioral changes, including high sensitivity to an N -methyl-D-aspartate (NMDA) receptor antagonist, hypoactivity in a novel environment, and decreased preference for social novelty, which are similar to those exhibited by human patients with schizophrenia. Thus, the CRISPR knock-out of the Gad1 rat could serve as a novel model for human schizophrenia. Cardiovascular disease is the leading cause of global death and threatens millions of people in their daily lives. Recently, Lu SX et al. (2020) Lu SX et al. (2020) knocked out heg1 , a gene encoding a heart development protein with epidermal growth factor (EGF) like domains 1, in zebrafish. The heg1 CRISPR zebrafish mutant developed severe cardiovascular malformations, including an abnormal heart rate, atrial ventricular enlargement, venous thrombosis, and slow blood flow. All these symptoms are similar to those associated with human heart failure and thrombosis ( Lu SX et al., 2020 Lu SX et al., 2020 ). Thus, the zebrafish heg1 CRISPR mutant has potential to serve as an animal model for analyzing the pathogenesis of cardiovascular diseases, and screening and testing new drugs and their potential therapeutic targets. As research progresses, more animal models will be generated using CRISPR/Cas technology, particularly newly developed prime editors and base editors that can easily target a single-base change. 4.2.2　Studying the pathogenesis and mechanisms associated with human diseases 3 Many genes are aberrantly expressed during the pathogenesis of human diseases and they are directly or indirectly associated with these diseases. However, the roles of many these genes during these processes are unclear. CRISPR/Cas genome editing provides a robust way to study the molecular mechanisms associated with pathogenesis, and can be used to obtain new therapeutic targets for disease treatment. In recent decades, cancers have been become the leading cause of death among all human diseases. Carcinogenesis is very complicated and usually involves genetic and epigenetic alterations in many genes and gene networks. Precise and efficient correction of these genes holds huge promise for cancer treatment. In the past eight years, CRISPR/Cas technology has been deeply explored to understand carcinogenesis and for cancer treatment, including impairing carcinogenesis-associated genes, exploring anticancer drugs, engineering immune cells and oncolytic viruses for enhancing cancer immunotherapeutics, and fighting oncogenic infections. By using an all-in-one lentiviral and retroviral delivery vectors hosting both Cas9 and sgRNAs, Malina et al. (2013) Malina et al. (2013) showed robust selection for the CRISPR-modified Trp53 locus following drug treatment. By linking Cas9 protein with green fluorescent protein (GFP), they also tracked disrupted Trp53 in chemoresistant lymphomas in the Eμ- myc mouse model. Chen et al. (2014) Chen et al. (2014) used a similar strategy to demonstrate that mixed lineage leukemia 3 ( MLL3 ) is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia (AML) in shNf1;Trp53 -/- primary mouse haematopoietic stem and progenitor cells (HSPCs). Xue et al. (2014) Xue et al. (2014) used CRISPR/Cas9 technology to directly target Pten and Trp53 tumor-suppressor genes in the mouse liver. They found that their CRISPR-generated Pten and p53 mutant induced liver tumors. The CRISPR/Cas-created mutation can be used to mimic the model phenotype caused by Cre‒ loxP -mediated deletion of Pten and p53 . These examples were among the first to show that CRISPR/Cas-based genome editing can be used to treat cancers and to study cancer pathogenesis. Since then, CRISPR/Cas genome editing has been quickly deployed to study mechanisms of disease occurrence, development, diagnosis, and therapeutic treatment. CRISPR/Cas-based genome and epigenome editing has been widely used to study the role of different genes, including oncogenes and tumor-suppressor genes, in various diseases, particularly in cancers. CRISPR/Cas was also developed as a new gene therapy tool to study and treat various human diseases, such as cancers. Programmed death-ligand 1 (PD-L1) is a regulatory molecule expressed in T cells and plays an important function in T cell-mediated immunotherapy. The PD-1/PD-L1 pathway serves as a critical immune checkpoint for many different cancers. CRISPR/Cas9 knock-out of the cdk5 gene blocked the expression of the PD-L1 gene and enhanced cell antitumor immunity ( Yahata et al., 2019 Yahata et al., 2019 ; Deng et al., 2020 Deng et al., 2020 ). CRISPR/Cas9 knock-out of cdk5 inhibited tumor cell growth in murine melanoma and lung metastasis suppression in triple-negative breast cancer ( Deng et al., 2020 Deng et al., 2020 ). CRISPR/Cas9 disruption of the expression of PD-L1 / PD-1 may also be beneficial for treating other cancers, including Epstein-Barr virus (EBV)-positive gastric cancer ( Su et al., 2017 Su et al., 2017 ), melanoma ( Mahoney et al., 2015 Mahoney et al., 2015 ), glioblastoma ( Choi et al., 2019 Choi et al., 2019 ), colorectal cancer ( Liao et al., 2020 Liao et al., 2020 ), and ovarian cancers ( Yahata et al., 2019 Yahata et al., 2019 ). Gao et al. (2021) Gao et al. (2021) used CRISPR/Cas genome editing to study the biological role of the AKT1 E17K mutation in a TP53-null background. Their results showed that AKT1 E17K inhibited cancer cell migration by abrogating β-catenin signaling. Using CRISPR/Cas9 genome editing, Bungsy et al. (2021) Bungsy et al. (2021) showed that the expression of RBX1 was associated with chromosome instability (CIN). A knock-out of the RBX1 gene significantly increased CIN phenotypes and increased Cyclin E1 levels and anchorage-independent growth in fallopian tube secretory epithelial cells, a cellular precursor of high-grade serous ovarian cancer (HGSOC) ( Bungsy et al., 2021 Bungsy et al., 2021 ). CRISPR/Cas can also be used as a screening tool for identifying genes associated with certain diseases, as well as a robust tool for drug screening. Soares et al. (2020) Soares et al. (2020) used CRISPR/Cas technology to screen and identify genes that confer susceptibility of AML to double negative T cell therapy. Their results suggested that CD64 is a predictive biomarker in AML patients. Takahashi et al. (2020) Takahashi et al. (2020) used a focused CRISPR/Cas9 screen with 3D culture models to target NRF2 and other redox regulatory genes, and revealed that hyperactive NRF2 serves as a prerequisite for spheroid cancer cell formation by regulating their proliferation and ferroptosis. By using targeted CRISPR/Cas9 dropout screening, Chen HR et al. (2021) Chen HR et al. (2021) identified methyltransferase-like 3 (METTL3) as the top essential m 6 A regulatory enzyme in colorectal cancer. Regulating gene expression (e.g., knock-out, knock-in, or gene regulation) by CRISPR/Cas can be used to increase target cell sensitivity to certain drugs, and can increase the efficiency of chemotherapy. CRISPR/Cas9 knock-out of the rev7 gene showed significant synergy with 5-fluorouracil (5-FU) and oxaliplatin in colorectal cancer in cell culture and in a murine xenograft model ( Gao et al., 2021 Gao et al., 2021 ). Sun et al. (2019) Sun et al. (2019) developed a drug-inducible CRISPR/Cas9 system that can be used both in vitro and in vivo for large-scale functional screening of human diseases. 4.2.3　Treating human genetic diseases including cancers and COVID-19 3 CRISPR/Cas has been developed as a powerful and promising tool for gene therapy ( Lotfi and Rezaei, 2020 Lotfi and Rezaei, 2020 ; Zeballos and Gaj, 2020 Zeballos and Gaj, 2020 ; Ferrari et al., 2021 Ferrari et al., 2021 ; Zhang, 2021 Zhang, 2021 ). There are many CRISPR/Cas studies on preclinical and clinical treatments of various diseases, including cancers and infectious diseases. In 2016, scientists at Sichuan University (Chengdu, China) and the Chengdu MedGenCell Co., Ltd., China, performed the first CRISPR/Cas gene therapy Phase 1 clinical trial, in which they knocked out the PD-1 gene in T cells for treating metastatic NSCLC using CRISPR/Cas9 genome editing technology. Since then, more CRISPR/Cas gene therapy clinical trials have been approved by different countries, especially in the USA, China, Germany, UK, Canada, Italy, France, Spain, and Australia. The clinically treated diseases varied widely, from different cancers to eye diseases and Rubinstein-Taybi syndrome, as well as human immunodeficiency virus (HIV) infection. Xu et al. (2019) Xu et al. (2019) reported their clinical trial (No. NCT03164135) involving treating HIV-infected patients with hematological malignances. They first used CRISPR/Cas genome editing technology to edit the CCR5 gene in HSPCs, and then transplanted the CRISPR-edited CCR5-ablated HSPCs into patients with HIV-1 infection and acute lymphoblastic leukemia. Their results showed that the CRISPR/Cas-edited donor cells persisted for more than 19 months after transplantation without causing gene-editing-related adverse events ( Xu et al., 2019 Xu et al., 2019 ; He, 2020 He, 2020 ). Stadtmauer et al. (2020) Stadtmauer et al. (2020) reported their results from a CRISPR/Cas9 gene therapy Phase 1 clinical trial on cancer treatment. First, they used CRISPR/Cas9 genome editing technology to knock-out TRAC , TRBC , and PDCD1 genes in T cells isolated from three individual patients. Then, they transferred the CRISPR/Cas-engineered T cells with a cancer-targeting gene, NY-ESO-1 , back into the patients. After nine months of treatment, all three patients showed promising results, demonstrating the high efficiency, technical safety, and feasibility of CRISPR/Cas genome editing technology for the treated cancers ( Stadtmauer et al., 2020 Stadtmauer et al., 2020 ). In another clinical trial, Lu Y et al. (2020) Lu Y et al. (2020) also demonstrated the safety and feasibility of using CRISPR/Cas9-engineered T cells to treat human cancers at advanced stages ( Lu Y et al., 2020 Lu Y et al., 2020 ). In their Phase 1 clinical trial (No. NCT02793856), they edited the PD-1 gene in T cells obtained from patients with advanced NSCLC. With a total of 22 patients enrolled, 12 received CRISPR gene therapy treatment with CRISPR-edited T cells for up to 96 weeks for treatment-related adverse events. Based on their study, after CRISPR gene therapy treatment of NSCLC, the median progression-free survival of 12 patients was 7.7 weeks, and the median overall survival was 42.6 weeks. No treatment-related deaths were observed in their clinical trials ( Lu Y et al., 2020 Lu Y et al., 2020 ). All these Phase 1 clinical trials demonstrated the safety and feasibility of CRISPR/Cas-based gene therapy using engineered immune T cells with a low off-target impact. Many other CRISPR/Cas gene therapy trials are underway, especially Phase 1 clinical trials ( Zhang, 2021 Zhang, 2021 ). As current CRISPR/Cas genome editing technology improves, a new generation of CRISPR/Cas genome editors, such as base editors and prime editors, will emerge, as well as new methods for delivering CRISPR/Cas reagents. Thus, the safety (e.g., lack of off-target impacts and low toxicity) and feasibility of the technology will be further improved. 5 CRISPR/Cas is a robust and powerful tool for precision breeding and crop improvement 1 Another major application of CRISPR/Cas genome editing technology is to improve crop yield and quality, as well as tolerance to various environmental stresses ( Tan YY et al., 2020 Tan YY et al., 2020 ; Zhang et al., 2021 Zhang et al., 2021 ). Since it was adopted in plants for genome editing, this technology has revolutionized studies in the field of plant molecular biology and is switching traditional plant breeding to precision breeding. 5.1 CRISPR/Cas systems have been quickly developed in a diversity of plant species 2 Since it was recognized as a naturally occurring genome editing tool, the CRISPR/Cas system has attracted increasing attention from the plant science community and was quickly applied to plant gene function studies and crop improvement. Shan et al. (2013) Shan et al. (2013) reported for the first time the establishment of a CRISPR/Cas system in two of the most widely cultivated food crops, wheat and rice. Since then, CRISPR/Cas genome editing technology has been widely established in almost all agriculturally important crops, including cotton, maize, soybean, and potato, as well as biofuel crops, such as switchgrass ( Table 3 Table 3 ). * There are many reports on different plant species for CRISPR/Cas-based genome editing. We attempted to list the first reports on each crop. If we missed some important studies, we apologize to the authors. CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; sgRNA: single guide RNA; miRNA: microRNA; Xcc : Xanthomonas citri subsp. citri . Common name Scientific name Transformation CRISPR/Cas Targeted gene Promotor for sgRNA Knock-out efficiency Off-target Reference Wheat Triticum aestivum Protoplast transformation, gene gun CRISPR/Cas9 TaMLO U6 Shan et al., 2013 Shan et al., 2013 Rice Oryza sativa Protoplast transformation, gene gun CRISPR/Cas9 OsPDS, OsBADH2 , Os02g23823 , and OsMPK2 U3 Varied, >15% Not found Shan et al., 2013 Shan et al., 2013 Maize Zea mays Protoplast transformation, Agrobacterium transformation CRISPR/Cas9 ZmIPK U3 13.1% Liang et al., 2014 Liang et al., 2014 Soybean Glycine max Agrobacterium rhizogenes transformation CRISPR/Cas9 GFP Glyma07g14530 , DDM1 , and miRNA U6 Varied, up to 95% Low Jacobs et al., 2015 Jacobs et al., 2015 Cotton Gossypium hirsutum Agrobacterium mediated CRISPR/Cas9 MYB U6 Not found Li et al., 2017 Li et al., 2017 Sorghum Sorghum bicolor Agrobacterium -mediated embryo transformation CRISPR/Cas9 DsRED2 U6 Jiang et al., 2013 Jiang et al., 2013 Sweet potato Ipomoea batatas Agrobacterium mediated CRISPR/Cas9 IbGBSSI and IbGBSSII U6 62%‒92% Wang HX et al., 2019 Potato Solanum tuberosum Agrobacterium mediated CRISPR/Cas9 StIAA2 StU6 Wang et al., 2015 Wang et al., 2015 Tomato Solanum lycopersic Agrobacterium -mediated transformation CRISPR/Cas9 GFP and SlSHR U6 Ron et al., 2014 Ron et al., 2014 Yam Dioscorea alata Agrobacterium mediated CRISPR/Cas9 PDS DaU6 83.3% Syombua et al., 2020 Cassava Manihot esculenta Agrobacterium mediated CRISPR/Cas9 MePDS AtU6 90%‒100% Odipio et al., 2017 Odipio et al., 2017 Switchgrass Panicum virgatum Agrobacterium mediated CRISPR/Cas9 Pv4cl1 U3 Park et al., 2017 Park et al., 2017 Switchgrass Panicum virgatum Agrobacterium mediated CRISPR/Cas9 tb1a , tb1b , and pgm U6 Varied, up to 95.5% Liu Y et al., 2018 Liu Y et al., 2018 Rapeseed Brassica napus Agrobacterium mediated CRISPR/Cas9 Alc U6 High Not detected Braatz et al., 2017 Braatz et al., 2017 Jatropha Jatropha curcas Agrobacterium mediated CRISPR/Cas9 JcCYP735A U3 Cai et al., 2018 Cai et al., 2018 Apple Malus prunifolia Agrobacterium mediated CRISPR/Cas9 PDS AtU6 31.8% Nishitani et al., 2016 Sweet orange Citrus sinensis Xcc -facilitated agroinfiltration CRISPR/Cas9 PDS 3.2%‒3.9% Jia and Wang, 2014 Jia and Wang, 2014 Pear Pyrus communis Agrobacterium mediated CRISPR/Cas9 PDS and TFL1 U3 and U6 Charrier et al., 2019 Poplar Populus Agrobacterium mediated CRISPR/Cas9 4CL U6 25% Zhou et al., 2015 Zhou et al., 2015 Sweet basil Ocimum basilicum Agrobacterium mediated CRISPR/Cas9 ObDMR1 Varied Navet and Tian, 2020 Navet and Tian, 2020 5.2 CRISPR/Cas systems have been widely used for improving crop resistance to biotic and abiotic stresses 2 Abiotic and biotic stresses are two major factors limiting crop growth and development as well as yield and quality. Particularly during climate change and industrialization, both stresses threaten natural plant development and agricultural practices. Although plants have evolved certain mechanisms to handle these stresses, and people have made significant progress on improving crop tolerance using both traditional breeding and transgenic technologies, there are still a big gap and a challenge for agricultural practices. Since it was adopted in plants, CRISPR/Cas-based genome editing technology has opened a new era for precision breeding for improved plant tolerance to abiotic and biotic stresses. With the increasing development and application of new technologies, such as next generation high-throughput deep sequencing technology, more and more genes, including both protein-coding and non-coding genes, have been identified which respond to different environmental stresses. Among these genes, some offer plant tolerance to these stresses, but there are many genes that make plants more sensitive to these stresses. For example, MILDEW-RESISTANCE LOCUS O ( MLO ) genes are widely considered to be plant susceptibility genes that are highly expressed in plants sensitive to powdery mildew, a widespread fungal disease in plants. MLOs play a negative role in the plant's response to powdery mildew infection. Loss-of-function of MLOs allows plants to gain resistance to powdery mildew disease in several important crops, including barley ( Büschges et al., 1997 Büschges et al., 1997 ). Thus, the manipulation of susceptibility genes (also called S-genes) has high potential in agricultural practices to increase crop tolerance to various diseases, reduce chemical pesticide usage, and protect the environment ( Filiz and Vatansever, 2018 Filiz and Vatansever, 2018 ). Inhibiting the expression of susceptibility genes is a great strategy for breeding new cultivars with high tolerance or even resistance to certain diseases. However, using traditional breeding technology and even transgenic technology to remove these genes is very hard, inefficient, and time- and lab-consuming. Although virus-induced gene silencing can be used to inhibit gene expression, it is difficult to completely inhibit the targeted genes and there are many uncertain outcomes. Thus, since scientists approved the application of CRISPR/Cas genome editing tools in plants, the technology has attracted lots of attention from scientific communities and biotechnological industries. In the past eight years, there have been many reports of creating genome-edited plants with resistance to viral, bacterial, and fungal diseases in different plant species using CRISPR/Cas technology ( Zhang et al., 2021 Zhang et al., 2021 ). These include CRISPR/Cas9 knock-outs of the following genes: mlo for plant resistance to powdery mildew in wheat ( Wang et al., 2014 Wang et al., 2014 ), tomato ( Nekrasov et al., 2017 Nekrasov et al., 2017 ; Martínez et al., 2020 Martínez et al., 2020 ) and grapevine ( Wan et al., 2020 Wan et al., 2020 ), pmr4 for plant resistance to powdery mildew in tomato (Santillán Martínez et al., 2020), 14-3-3 gene for resistance to Verticillium dahlia in cotton ( Zhang ZN et al., 2018 Zhang ZN et al., 2018 ), crt1a for resistance to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape ( Pröbsting et al., 2020 Pröbsting et al., 2020 ), OsERF922 for resistance to Magnaporthe oryzae in rice ( Wang et al., 2016 Wang et al., 2016 ), Clpsk1 for resistance to Fusarium oxysporum f. sp. niveum in watermelon ( Zhang et al., 2020 Zhang et al., 2020 ), eif4e for resistance to Cucumber vein yellowing virus , Zucchini yellow mosaic virus , and Papaya ring spot mosaic virus‐W in cucumber ( Chandrasekaran et al., 2016 Chandrasekaran et al., 2016 ), CsWRKY22 for resistance to Xanthomonas citri subsp. citri ( Xcc ) in orange ( Wang LJ et al., 2019 Wang LJ et al., 2019 ), CsLOB1 for resistance to Xcc in citrus ( Peng et al., 2017 Peng et al., 2017 ), SlJAZ2 for resistance to Pseudomonas syringae pv. tomato ( Pto ) DC3000 in tomato ( Ortigosa et al., 2019 Ortigosa et al., 2019 ), Os8N3 for resistance to Xanthomonas oryzae pv. oryzae ( Xoo ) in rice ( Kim et al., 2019 Kim et al., 2019 ), and LIPOXYGENASE 3 ( lox3 ) for resistance to Ustilago maydis in maize ( Pathi et al., 2020 Pathi et al., 2020 ). Compared with pathogen infection, fewer susceptibility genes have been identified in plants in response to abiotic stresses. Thus, progress has been much slower in creating CRISPR/Cas genome-edited lines for plant resistance to various abiotic stresses, such as drought, salinity, extreme temperature (cold and heat), and environmental pollution. However, as more negative genes have been identified, more progress has been made in creating genome-edited plants with high tolerance to abiotic stresses. During long-term studies, scientists found that many structural and regulatory genes are associated with a plant's response to different environmental abiotic stresses. Many genes controlling plant development, particularly root development, also contribute to the plant's response to abiotic stresses. For example, arginase and nitric oxide synthase (NOS) are two important enzymes regulating nitric oxide (NO) synthesis and root development, particularly lateral root development ( Correa-Aragunde et al., 2004 Correa-Aragunde et al., 2004 ). These two enzymes compete for arginine which is important for NO biosynthesis. Reduced arginase activity increased NO accumulation in Arabidopsis arginase gene ( arg ) mutants and enhanced lateral and adventitious root development ( Flores et al., 2008 Flores et al., 2008 ). In contrast, overexpression of the arg gene inhibited NO accumulation and further repressed lateral root development in transgenic cotton ( Meng et al., 2015 Meng et al., 2015 ). Using CRISPR/Cas9 genome editing technology, Wang et al. (2017) Wang et al. (2017) successfully knocked out the arg gene in cotton. This significantly increased lateral root differentiation and development as shown by an increase in root number of more than 25%, and of root surface area by more than 50%, in both low- and high-nitrogen conditions ( Wang et al., 2017 Wang et al., 2017 ). This suggests that CRISPR/Cas9-edited arg knock-out plants have better root development, enhancing plant growth and tolerance to different abiotic stresses, including nitrogen deficiency and drought stress ( Zhang et al., 2021 Zhang et al., 2021 ; Peng et al., 2021 Peng et al., 2021 ). Knock-out of the G protein genes, gs3 and dep1 , significantly improved rice tolerance to different abiotic stresses, including drought, chilling, and salinity stresses. Under salinity treatment, all tested genome-edited lines showed enhanced tolerance compared to the controls ( Cui et al., 2020 Cui et al., 2020 ). Abiotic stresses induced aberrant expression of many transcription factors and non-coding RNAs that play important roles in the plant's response to these environmental stresses. There are many classes of transcription factors that regulate lots of downstream genes for controlling plant growth, development, and response to various environmental stresses. Knock-out or overexpression of these transcription factor genes alters the expression profiles of many other genes and then affects plant development and response to different stresses. Auxin response factor (ARF) is one family of functionally distinct DNA-binding transcription factors found widely in all plant species ( Li SB et al., 2016 Li SB et al., 2016 ). CRISPR/Cas9 knock-out of the arf4 transcription factor gene improved plant tolerance to salinity and osmotic stresses in tomato ( Bouzroud et al., 2020 Bouzroud et al., 2020 ). Using the CRISPRa epigenome editor, Roca Paixão et al. (2019) Roca Paixão et al. (2019) successfully fused dCas9 protein with a histone acetyltransferase (AtHAT1) and used this fused CRISPR/dCas9 system to target the abscisic acid (ABA)-responsive element-binding protein 1 (AREB1)/ABRE-binding factor 2 (ABF2). Their results showed that the CRISPRa dCas9 HAT system activated the endogenous promoter of AREB1 and enhanced plant tolerance to drought stress in Arabidopsis . Under salinity stress, the overexpression technology combined with the CRISPR-Cas9 system demonstrated that the transcription factor gene NAC06 caused proline and glycine accumulation to alleviate or avoid reactive oxygen species (ROS)-induced oxidative stress and maintained ionic homeostasis and Na + /K + ratio in soybean hairy roots. Consequently, soybean plant tolerance to salinity stress was enhanced ( Li et al., 2021 Li et al., 2021 ). 5.3 CRISPR/Cas systems have been widely used in improving crop yield and quality 2 Improving crop yield and quality is the ultimate goal for precision crop breeding and can be achieved directly or indirectly, including improving crop tolerance to various environmental stresses. Crop yield is generally controlled by multiple genes, and single genes that significantly control crop yield alone are hard to find. Although great progress has been achieved in obtaining transgenic plants, the main focus has been on insect-resistance and herbicide tolerance. These transgenic crops, particularly insect-resistant biotech crops (such as Bt cotton), have brought huge economic and social benefits, and in certain cases the crop yield has also significantly increased because of reduced losses caused by different pests. However, as more modern techniques, such as high-throughput deep sequencing, have been used to investigate the molecular mechanisms controlling crop yield, more genes associated with crop yield have been identified. Among them, there is one class of genes, called negative regulators, that negatively affect crop yield. These negative regulators of crop yield, just like S genes in the plant's response to pathogen infection, provide a great potential target for CRISPR/Cas to improve crop yield ( Table 4 Table 4 ). Gn1a , dep1 , and gs3 are three negative regulators associated with grain number per panicle, grain size, and seed size in rice ( Ashikari et al., 2005 Ashikari et al., 2005 ). Gn1a is a gene for cytokinin oxidase/dehydrogenase (OsCKX2). Reduced expression of gn1a increased the number of reproductive organs and enhanced grain yield ( Ashikari et al., 2005 Ashikari et al., 2005 ). Li MR et al. (2016) Li MR et al. (2016) used CRISPR/Cas9 technology to successfully knock out all three of these genes. Their CRISPR/Cas9 genome-edited rice showed enhanced grain number, dense erect panicles, and larger grain size. It is also well known that grain width 2 ( gw2 ), gw5 , and thousand-grain weight 6 ( tgw6 ) negatively regulate grain weight ( Zuo and Li, 2014 Zuo and Li, 2014 ; Xu et al., 2016 Xu et al., 2016 ). Knock-out of these three genes significantly increased grain weight in rice, and simultaneous knock-out of two or more genes resulted in a larger grain size and crop yield ( Xu et al., 2016 Xu et al., 2016 ). Also, CRISPR knock-out of the OsPAO5 gene enhanced the grain weight, grain number, and yield potential in rice ( Lv et al., 2021 Lv et al., 2021 ). In the biofuel crop switchgrass, CRISPR/Cas9 knock-out of the teosinte branched 1 ( tb1 ) gene increased the number of plant tillers and fresh biomass ( Liu et al., 2020 Liu et al., 2020 ). In rapeseed, simultaneous knock-out of all four BnaMAX1 alleles resulted in semi-dwarf and increased branching phenotypes with more siliques. These traits contributed to increased yield compared with their wild-type controls ( Zheng et al., 2020 Zheng et al., 2020 ). There are three GW2 homeologs (TaGW2-A1, -B1, and -D1) in hexaploid wheat. CRISPR/Cas9 knock-out of an individual homeolog affected wheat grain width and length and thousand-grain weight and yield. Double CRISPR knock-out of TaGW2 showed a stronger effect on these traits than a single mutation ( Zhang Y et al., 2018 Zhang Y et al., 2018 ). By targeted editing of the early heading date 1 ( Ehd1 ) gene by CRISPR/Cas9, Wu et al. (2020) Wu et al. (2020) generated both frame-shift and in-frame deletion mutants in four rice cultivars. The mutants showed significantly longer basic vegetative growth periods and significantly improved yield potential compared with wild types when planted at low-latitude stations. CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein. Crop species Targeted gene Improved trait CRISPR/Cas editor Reference Rice gn1a , dep1 , and gs3 Grain number, dense erect panicles, and larger grain size CRISPR/Cas9 Li MR et al., 2016 Li MR et al., 2016 Rice gw2 , gw5 , and tgw6 Grain size and crop yield CRISPR/Cas9 Xu et al., 2016 Xu et al., 2016 Rice OsPAO5 Grain weight, grain numbers, and yield CRISPR/Cas9 Lv et al., 2021 Lv et al., 2021 Switchgrass tb1 Tillers and fresh biomass CRISPR/Cas9 Liu et al., 2020 Liu et al., 2020 Rapeseed BnaMAX1 Plant architecture and yield CRISPR/Cas9 Zheng et al., 2020 Zheng et al., 2020 Soybean GmLHY Plant height and internode length CRISPR/Cas9 Cheng et al., 2019 Cheng et al., 2019 Soybean AP1 Flowering time and plant height CRISPR/Cas9 Chen et al., 2020 Chen et al., 2020 Soybean GmNMHC5 Flowering and maturity CRISPR/Cas9 Wang WT et al., 2020 Wang WT et al., 2020 Wheat TaGW2 Grain weight and protein content CRISPR/Cas9 Zhang Y et al., 2018 Zhang Y et al., 2018 Rice OsGcs1 High-quality sugar production CRISPR/Cas9 Honma et al., 2020 Honma et al., 2020 Rice Ehd1 Basic vegetative growth CRISPR/Cas9 Wu et al., 2020 Wu et al., 2020 Sugar is an important component in our daily life and in biofuel production. However, sugarcane and sugar beet are the only two crops that produce significant amounts of sugar. Recently, Honma et al. (2020) Honma et al. (2020) knocked out the OsGcs1 gene in rice by CRISPR/Cas and obtained sugary-rice grains containing a high percentage of high-quality sugar. The CRISPR/Cas-edited rice ovules contained 10%–20% sugar with an extremely high sucrose content (98%) ( Honma et al., 2020 Honma et al., 2020 ). This may provide a new way to develop novel sugar-producing plants. Individual components, such as amino acids or carbohydrates, significantly affect crop quality. Thus, targeting the specific biosynthetic pathway of an individual component is a promising strategy to improve crop quality. Resistant starch and amylose benefit human health by lowering the potential risk of certain serious diseases. Improving the content of resistant starch and amylose is a long-standing goal for breeders and scientists. By using CRISPR/Cas9 genome editing technology, Li JY et al. (2020) Li JY et al. (2020) knocked out the TaSBEIIa gene in both winter and spring wheat cultivars. The genome-edited wheat contained a significantly higher amount of amylose, resistant starch, protein, and soluble pentosan than the wild type ( Li JY et al., 2020 Li JY et al., 2020 ). By knocking out the starch branching enzyme genes, SBEI and SBEIIb , Sun et al. (2017) Sun et al. (2017) obtained transgene-free rice with an amylose content as high as 25.0% and a resistant starch content of 9.8%. By using CRISPR/Cas9 genome editing, Zhang JS et al. (2018) Zhang JS et al. (2018) knocked out the Waxy gene in two widely cultivated elite japonica rice cultivars. Their results showed that Waxy genome-edited rice contained lower amylose and converted the rice into glutinous rice without affecting other desirable agronomic traits. Gluten is a family of storage proteins, which plays an important role in cooking, particularly for making bread and noodles, the most commonly consumed foods. Gluten is widely found in certain cereal grains, including wheat, barley, and rye. However, many people are sensitive or even allergic to gluten, and show celiac disease. Thus, breeding gluten-free crops is necessary and important. In breeding programs, scientists and breeders have used traditional breeding technology and mutation breeding (e.g., chemically or physically induced amastigogenesis) to obtain low-gluten-content cultivars (van den Broeck et al., 2009; Juhász et al., 2020 Juhász et al., 2020 ). Although some progress has been made, the traditional methods are time-, lab-, and cost-intensive and usually require the generation of a huge breeding population and from which to select plants with the desired traits. As scientists understand more about gluten biosynthesis and the development of RNA interference (RNAi) technology, by targeting a specific gene during gluten biosynthesis, several different laboratories have knocked down an individual gene and obtained RNAi plants with a reduced gluten content ( Gil-Humanes et al., 2008 Gil-Humanes et al., 2008 , 2010 2010 ; Becker et al., 2012 Becker et al., 2012 ; Barro et al., 2016 Barro et al., 2016 ; Altenbach et al., 2019 Altenbach et al., 2019 ). However, RNAi technology cannot completely eliminate gluten biosynthesis in plants, and in certain cases this technology is hard to handle due to its instability. The rapid development of CRISPR/Cas systems provides a perfect technology for controlling gluten biosynthesis, and opens a new era for breeding gluten-free cultivars. By targeting the α-gliadin genes, Sánchez-León et al. (2018) Sánchez-León et al. (2018) successfully obtained low-gluten transgene-free wheat using CRISPR/Cas9 genome editing technology. All 21 CRISPR mutant lines showed a significant reduction (up to 85%) in gluten content. Using the same technology, another research group targeted both α‍- and γ‍-gliadins and observed clear changes in the gluten profiles of CRISPR/Cas9 genome-edited bread wheat lines ( Jouanin et al., 2019 Jouanin et al., 2019 ). There are many different types of plant oils, and different oils have different quality. Generally speaking, oleic oil has better quality because of its high content of healthier fats (monounsaturated and polyunsaturated) ( Zhou et al., 2020 Zhou et al., 2020 ). Thus, scientists have been attempting to modify oil components to improve oil quality by using advanced CRISPR/Cas genome editing. The fatty acid desaturase 2 (FAD2) enzyme is the enzyme that controls the biological switch between oleic acid and linoleic acid ( Dar et al., 2017 Dar et al., 2017 ). Knock-out of fad2 genes significantly increases oleic acid content and improves oil quality. Thus, fad2 genes have been become a target gene for using CRISPR/Cas to modify oil quality in different plant species. By targeting the fad2 gene using CRISPR/Cas9, Jiang et al. (2017) Jiang et al. (2017) obtained CRISPR/Cas genome-edited Camelina sativa plants with oleic acid content increased from 16% to ˃50% of the fatty acid composition ( Jiang et al., 2017 Jiang et al., 2017 ). They also observed that CRISPR/Cas genome-edited fad2 plant seeds had a significantly reduced content of the less desirable polyunsaturated fatty acids, linoleic acid (a decrease from about 16% to < 4%) and linolenic acid (a decrease from about 35% to < 10%). fad2 gene was also knocked out by CRISPR/Cas in other plant species, including rice ( Abe et al., 2018 Abe et al., 2018 ), rapeseed ( Huang et al., 2020 Huang et al., 2020 ), tobacco ( Tian et al., 2020 Tian et al., 2020 ), cotton ( Chen YZ et al., 2021 Chen YZ et al., 2021 ), soybean (al Amin et al., 2019), and peanut ( Yuan et al., 2019 Yuan et al., 2019 ), to obtain high-quality oil with high oleic/low linoleic acid content. In tobacco, CRISPR/Cas9 knock-out of the fad2 gene dramatically increased the oleic acid content from 11% to ˃79%, whereas linoleic acid was reduced from 72% to 7% ( Tian et al., 2020 Tian et al., 2020 ). Many plant oils contain significant amounts of long-chain fatty acids that are undesirable for many different purposes. Using CRISPR/Cas9, Ozseyhan et al. (2018) Ozseyhan et al. (2018) successfully knocked out the fatty acid elongase 1 ( FAE1 ) gene in C. sativa . Their results showed that C20–C24 very long-chain fatty acids (VLCFAs) were reduced to less than 2% of the total fatty acids from over 22% in the wild type ( Ozseyhan et al., 2018 Ozseyhan et al., 2018 ). CRISPR/Cas genome editing technology has also been used to modify lipid content quality. For example, Lin and Ng (2020) Lin and Ng (2020) used CIRSPR/Cas9 to knock out the fad3 gene and obtained a 46% higher accumulation of lipids in Chlorella vulgaris FSP-E. In bovine mammary epithelial cells, CRISPR/Cas knock-out of the butyrophilin subfamily 1 member A1 ( BTN1A1 ) gene changed the lipid droplet formation and phospholipid composition. The percentage of phosphatidylethanolamine (PE) increased, while the percentage of phosphatidylcholine (PC) decreased, which resulted in a lower PC/PE ratio ( Han et al., 2020 Han et al., 2020 ). CRISPR/Cas9 knock-out of the fad2 gene in Atlantic salmon also altered their lipid metabolism ( Jin et al., 2020 Jin et al., 2020 ). During long evolutionary history, plants have evolved certain pathways to synthesize specific components, some of which are good for human health, while others are not. For example, red rice contains high levels of proanthocyanidins and anthocyanins that are health-promoting nutrients. The red pericarp found widely in cultivated rice ancestors and wild rice species is controlled by two complementary genes, Rc and Rd ( Furukawa et al., 2007 Furukawa et al., 2007 ). However, during rice evolution and domestication, a 14-bp frame-shift deletion in the Rc gene enabled the selection of white rice ( Sweeney et al., 2007 Sweeney et al., 2007 ). In a recent study, Zhu et al. (2019) Zhu et al. (2019) used CRISPR/Cas9 genome editing technology to successfully restore the function of the Rc gene by reverting the 14-bp frame-shift deletion to in-frame mutations ( Zhu et al., 2019 Zhu et al., 2019 ). Using this strategy, they successfully converted three elite white pericarp rice cultivars into red pericarp types, all of which can accumulate high levels of proanthocyanidins and anthocyanins in their red grains without affecting other important agronomic traits ( Zhu et al., 2019 Zhu et al., 2019 ). GABA is a non-proteinogenic amino acid, which has beneficial human health effects. Increasing the GABA content of fruit enhances its health benefit by lowering blood pressure. GABA biosynthesis is controlled mainly by GAD. By targeting SlGAD2 and GlGAD3 , CRISPR/Cas9-edited tomato plants accumulated 7- to 15-fold more GABA in their fruits ( Nonaka et al., 2017 Nonaka et al., 2017 ). By targeting genes associated with GABA metabolism, including GABA‐TP1 , GABA‐TP2 , GABA‐TP3 , CAT9 , and SSADH , Li et al. (2018b) Li et al. (2018b) obtained CRISPR genome-edited tomatoes with GABA content up to 19-fold higher than that of the wild type. Hunziker et al. (2020) Hunziker et al. (2020) used the CRISPR/Cas9 system fused with target activation-induced cytidine deaminase (Target-AID) base-editing technology to knock out the SlDDB1 , SlDET1 , and SlCYC-B genes to alter carotenoid accumulation. Lycopene is a plant nutrient with antioxidant properties which have been linked to beneficial effects on several diseases, including heart and cardiovascular diseases and certain cancers. Increasing lycopene content in fruits has significant benefits. By targeting genes associated with lycopene biosynthesis, such as SGR1 (GenBank accession No. DQ100158), lycopene ε-cyclase ( LCY-E ; GenBank accession No. EU533951), β-lycopene cyclase ( Blc ; GenBank accession No. XM_010313794), lycopene β-cyclase 1 ( LCY-B1 ; GenBank accession No. EF650013), and LCY-B2 (GenBank accession No.AF254793), Li XD et al. (2018) Li XD et al. (2018) obtained CRISPR-edited tomatoes with up to a 5.1-fold increase in lycopene content using a bidirectional strategy: promoting the biosynthesis of lycopene, while inhibiting its conversion to β- and α-carotene. Using similar technology, Kaur et al. (2020) Kaur et al. (2020) obtained β-carotene-enriched Cavendish bananas with β-carotene content increased by up to 6-fold (about 24 μg/g) compared with unedited plants. CRISPR/Cas genome editing is also being used to improve crop storage and post-harvest quality ( Table 5 Table 5 ). The storage and transportation of certain fruits, such as tomato and peach, are long-standing problems. When fruits are fully mature with great taste, they become soft and difficult to store long-term and transport. Yu et al. (2017) Yu et al. (2017) used CRISPR/Cas genome editing technology to obtain both ALC gene mutagenesis and replacement in tomato. As in other plant species, CRISPR HDR-mediated gene replacement is much more difficult than CRISPR/Cas knock-out mutagenesis ( Yu et al., 2017 Yu et al., 2017 ). The CRISPR-edited tomato demonstrated improved storage performance and long-shelf life without affecting other agronomic traits, such as plant size and fruit firmness ( Yu et al., 2017 Yu et al., 2017 ). Using a CRISPR/Cas9 knock-out of a tomato ripening-related lncRNA, lnRNA1459 , Li R et al. (2018a) Li R et al. (2018a) obtained tomato plants with altered fruit ripening in tomato. CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; AID: activation-induced cytidine deaminase; NHEJ: non-homologous end joining; HDR: homology-directed repair. Crop species 2 Targeted gene 3 Improved trait CRISPR/Cas editor Reference Carbohydrate 2 3 Wheat 2 TaSBEIIa 3 Amylose and resistant starch content CRISPR/Cas9 Li JY et al., 2020 Li JY et al., 2020 Rice 2 SBEI and SBEIIb 3 Amylose and resistant starch content CRISPR/Cas9 Sun et al., 2017 Sun et al., 2017 Rice 2 Waxy 3 Amylose content CRISPR/Cas9 Zhang JS et al., 2018 Zhang JS et al., 2018 Potato 2 GBSS 3 Amylose content CRISPR/Cas9 Andersson et al., 2018 Andersson et al., 2018 Potato 2 GBSS1 3 Amylose content CRISPR/Cas9 Kusano et al., 2018 Kusano et al., 2018 Protein 2 3 Wheat 2 α‐gliadin 3 Low-gluten CRISPR/Cas9 Sánchez-León et al., 2018 Sánchez-León et al., 2018 Wheat 2 α‐gliadin and γ-gliadin 3 Low-gluten CRISPR/Cas9 Jouanin et al., 2019 Jouanin et al., 2019 Oil content and quality 2 3 Camelina sativa 2 fad2 genes 3 Oil quality CRISPR/Cas9 Jiang et al., 2017 Jiang et al., 2017 Rice 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Abe et al., 2018 Abe et al., 2018 Peanut 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Yuan et al., 2019 Yuan et al., 2019 Rapeseed 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Huang et al., 2020 Huang et al., 2020 Rapeseed 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Okuzaki et al., 2018 Okuzaki et al., 2018 Tobacco 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Tian et al., 2020 Tian et al., 2020 Cotton 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 Chen YZ et al., 2021 Chen YZ et al., 2021 Soybean 2 fad2 genes 3 High oleic/low linoleic acid CRISPR/Cas9 al Amin et al., 2019 al Amin et al., 2019 Camelina sativa 2 fae1 genes 3 Oil quality CRISPR/Cas9 Ozseyhan et al., 2018 Ozseyhan et al., 2018 Camelina sativa 2 CsDGAT1 and CsPDAT1 3 Seed oil production and fatty acid composition CRISPR/Cas9 Aznar-Moreno and Durrett, 2017 Aznar-Moreno and Durrett, 2017 Camelina sativa 2 CsCRUC 3 Seed protein and oil profile CRISPR/Cas9 Lyzenga et al., 2019 Lyzenga et al., 2019 Rapeseed 2 BnPAT and BnLPAT 3 Oil and starch content CRISPR/Cas9 Zhang et al., 2019 Zhang et al., 2019 Rapeseed 2 BnTT8 3 Oil and protein CRISPR/Cas9 Zhai et al., 2020 Zhai et al., 2020 Rapeseed 2 BnITPK 3 Phytic acid content CRISPR/Cas9 Sashidhar et al., 2020 Sashidhar et al., 2020 Rapeseed 2 BnSFAR4 and BnSFAR5 3 Seed oil content CRISPR/Cas9 Karunarathna et al., 2020 Karunarathna et al., 2020 Rapeseed 2 BnTT2 3 Oil and atty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3) CRISPR/Cas9 Xie T et al., 2020 Xie T et al., 2020 Wheat 2 TaGW2 3 Grain weight and protein content CRISPR/Cas9 Zhang Y et al., 2018 Zhang Y et al., 2018 Anti-nutrient 2 3 Grape 2 IdnDH 3 Reduced tartaric acid content CRISPR/Cas9 Ren et al., 2016 Ren et al., 2016 Potato 2 St16DOX 3 Reduced steroidal glycoalkaloid content CRISPR/Cas9 Nakayasu et al., 2018 Nakayasu et al., 2018 Maize 2 ZmIPK 3 Reduced phytic acid content CRISPR/Cas9 Liang et al., 2014 Liang et al., 2014 Functional metabolites 2 3 Rice 2 rc 3 Roanthocyanidin and anthocyanin content CRISPR/Cas9 Zhu et al., 2019 Zhu et al., 2019 Potato 2 StPPO2 3 Enzymatic browning CRISPR/Cas9 González et al., 2020 González et al., 2020 Tomato 2 ANT1 3 Anthocyanin content CRISPR/Cas9 Čermák et al., 2015 Čermák et al., 2015 Tomato 2 SlGAD2 and GlGAD3 3 γ-Aminobutyric acid (GABA) content CRISPR/Cas9 Nonaka et al., 2017 Nonaka et al., 2017 Tomato 2 GABA‐TP1 , GABA‐TP2 , GABA‐TP3 , CAT9 , and SSADH 3 GABA content CRISPR/Cas9 Li R et al., 2018b Li R et al., 2018b 2 Tomato 2 SlDDB1 , SlDET1 , and SlCYC-B Carotenoid accumulation 2 CRISPR/Cas9-AID base editor Hunziker et al., 2020 Hunziker et al., 2020 2 Tomato 2 SGR1 , LCY-E , Blc , LCY-B2 , and LCY-B1 Lycopene content 2 CRISPR/Cas9 Li XD et al., 2018 Li XD et al., 2018 2 Tomato 2 CycB , FW2.2 , etc. Lycopene content 2 CRISPR/Cas9 Zsögön et al., 2018 Zsögön et al., 2018 2 Tomato 2 GGP1 , GLV3 , etc. Vitamin C content 2 CRISPR/Cas9 Li TD et al., 2018 Li TD et al., 2018 2 Banana 2 LCYε β-Carotene 2 CRISPR/Cas9 Kaur et al., 2020 Kaur et al., 2020 2 Chinese kale 2 BoaCRTISO Color-related chlorophyll and carotenoid content 2 CRISPR/Cas9 Sun et al., 2020 Sun et al., 2020 2 Rice 2 OsCYP97A4 , OsDSM2 , OsCCD4a , OsCCD4b , and OsCCD7 Carotenoid accumulation 2 CRISPR/Cas9 Yang et al., 2017 Yang et al., 2017 2 Other traits 2 2 2 Tomato 2 ALC Improved storage performance and long-shelf life 2 CRISPR/Cas9 (NHEJ and HDR) Yu et al., 2017 Yu et al., 2017 2 Tomato 2 LncRNA1459 Fruit ripening 2 CRISPR/Cas9 Li R et al., 2018a Li R et al., 2018a 2 Tomato 2 RIN Fruit ripening 2 CRISPR/Cas9 Ito et al., 2015 Ito et al., 2015 2 Tomato 2 LeMADS-RIN Fruit ripening 2 CRISPR/Cas9 Jung et al., 2018 Jung et al., 2018 6 Current challenges of CRISPR/Cas and its future directions 1 In the past decade, significant progress has been made in discovering, modifying, and adopting CRISPR/Cas systems in gene function studies, clinical research, and crop improvement. However, there are still several major issues associated with this advanced technology that need to be resolved before it will be widely used in biomedicine and precision breeding. 6.1 CRISPR/Cas delivery is still one bottleneck hindering its wider usage 2 Whether treating human genetic diseases or carrying out precision crop breeding, the first important step is to deliver CRISPR/Cas reagents into the target cells. In plants, CRISPR/Cas-based genome editing is highly dependent on plant tissue culture-based gene transformation. Currently, the most efficient way to obtain genome editing events is by using Agrobacterium -mediated gene transformation, which is limited to a small number of plant species. Although plants of some species, such as agriculturally important crops, can be regenerated, this is limited to only a few genotypes or cultivars. Thus, developing a highly efficient genotype-independent plant tissue culture and plant regeneration system, or developing a new transformation method avoiding plant regeneration, is urgently needed. It is possible to improve plant regeneration capacity through testing different explants, plant growth hormones/regulators, different active chemicals, and combinations of these effectors. At first, it was extremely difficult to obtain regenerated plants from rice and maize tissue culture. However, after many years of hard work, this problem was solved in rice and maize, mainly by selecting immature embryos as explants ( Peng et al., 2021 Peng et al., 2021 ). Now, rice has been become a model plant species for both genetic and applied research. One of the reasons is that there is now a highly efficient tissue culture and transformation system for rice. Cotton is also an important crop, and is thought to be one of the most difficult plant species for obtaining somatic embryogenesis and plant regeneration. However, modifying the plant growth medium and culture strategies, including starvation and drought treatment, significantly improved the capacity of cotton tissue culture and plant regeneration, and regenerated plants have now been obtained from almost all tested cotton genotypes ( Zhang et al., 2009 Zhang et al., 2009 ). Although we can obtain genome-edited plants through plant tissue culture-based methods, many issues, such as induced mutations during plant tissue culture and the lengthy process, have limited the application of CRISPR/Cas technology. Thus, developing a tissue culture-independent delivery method will be the best choice for CRISPR/Cas genome editing. Recently, Ma et al. (2020) Ma et al. (2020) used Sonchus yellow net rhabdovirus (SYNV) to successfully deliver both Cas9 protein and sgRNAs (transfer RNA (tRNA)–guide RNA (gRNA)–tRNA fusion) into plant cells, and obtained CRISPR/Cas genome editing events. Further modifications of this virus-based CRISPR/Cas reagent delivery system will provide a tissue culture-independent transgenic and genome editing system that can be used for any plant species without the need for a complicated laboratory process ( Liu and Zhang, 2020 Liu and Zhang, 2020 ). 6.2 Off-target effects are still a big challenge for clinical treatments 2 Off-target impacts are a big issue for CRISPR/Cas-based genome editing, particularly for clinical treatment that does not allow any errors. Although it is not so critical for genome editing in plants, it may affect precision breeding by affecting other agriculturally important traits. Off-targets occur in CRISPR/Cas-based genome editing, for all types of genome editors, particularly in animals. Although scientists have been attempting to limit off-target impacts, it seems that it is hard to eliminate them completely. Currently, there are several efficient strategies used for reducing CRISPR/Cas-based genome editing off-targets. (1) Selecting an appropriate CRISPR/Cas reagent delivery system. Different delivery systems may have significantly different effects, not only for genome editing efficiency and outcomes, but also in relation to federal regulations. No matter what delivery strategy, the Cas DNA/RNA/protein and sgRNAs need to be delivered into plant/animal cells. If Cas/sgRNA genes are inserted into the genome, this will cause those genes to be permanently expressed in the cells and cause potential off-target effects. However, if the Cas/sgRNA genes are not inserted into the plant/animal genome, and exist in the target cells for only a short period, the rate of off-target effects may be minimized. Many studies show that delivery of Cas-sgRNA ribonucleoprotein (RNP) complex into cells reduces off-target impacts mainly because the CRISPR/Cas9 system is not inserted into the host genome and the RNA/protein has a short life time ( Doudna, 2020 Doudna, 2020 ). (2) Designing high-fidelity sgRNA and selecting the right Cas enzymes and genome editing tools can also reduce the rate of off-target effects. If we can fuse a proofreading enzyme, like DNA polymerase, with the Cas enzyme, when an off-target event occurs, the proofreading enzyme will correct any errors ( Zhang and Zhang, 2020b Zhang and Zhang, 2020b ). This will eliminate the off-target impact of CRISPR/Cas genome editing. Another potential way to reduce off-target impact is to deliver the CRISPR/Cas/gRNA reagents only to the targeted cells. 6.3 CRISPR/Cas multiplex genome editing: good or bad? 2 In recent years, multiplex CRISPR/Cas genome editing has attracted attention from many research laboratories, and has been used to edit genes in certain plant and animal genomes. Although it can be used to edit multiple genes simultaneously, it also causes lots of potential problems, including more off-target impacts and removal of long DNA fragments. Thus, multiplex CRISPR/Cas genome editing is not a robust and high-tech technology, and adds only one or more gRNAs into the constructs. Higher off-target impacts and removal of long DNA fragments will cause more serious outcomes. Therefore, multiplex CRISPR/Cas genome editing does not yet have general practical application in clinical treatment or precision breeding. Even for gene function studies, it may take a lot more time to select and identify CRISPR/Cas mutations after genome editing. To reduce potential side effects, when using multiplex CRISPR/Cas genome editing technology the targeted sites should not be located on the same chromosome. This will help reduce the frequency of long DNA fragment deletions. References Abe K , Araki E , Suzuki Y , et al. , 2018 . Production of high oleic/low linoleic rice by genome editing . Plant Physiol Biochem , 131 : 58 - 62 . https://doi.org/10.1016/j.plaphy.2018.04.033 https://doi.org/10.1016/j.plaphy.2018.04.033 Production of high oleic/low linoleic rice by genome editing Addington AM , Gornick M , Duckworth J , et al. , 2005 . GAD 1 ( 2 q 31 . 1), which encodes glutamic acid decarboxylase (GAD 67 ), is associated with childhood-onset schizophrenia and cortical gray matter volume loss . Mol Psychiatry , 10 ( 6 ): 581 - 588 . https://doi.org/10.1038/sj.mp.4001599 https://doi.org/10.1038/sj.mp.4001599 GAD Adli M , 2018 . The CRISPR tool kit for genome editing and beyond . Nat Commun , 9 : 1911 . https://doi.org/10.1038/s41467-018-04252-2 https://doi.org/10.1038/s41467-018-04252-2 The CRISPR tool kit for genome editing and beyond Ai JW , Zhou X , Xu T , et al. , 2019 . CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis . Emerg Microbes Infect , 8 ( 1 ): 1361 - 1369 . https://doi.org/10.1080/22221751.2019.1664939 https://doi.org/10.1080/22221751.2019.1664939 CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis al Amin N , Ahmad N , Wu N , et al. , 2019 . CRISPR-Cas9 mediated targeted disruption of FAD2-2 microsomal omega-6 desaturase in soybean ( Glycine max . L). BMC Biotechnol , 19 : 9 . https://doi.org/10.1186/s12896-019-0501-2 https://doi.org/10.1186/s12896-019-0501-2 CRISPR-Cas9 mediated targeted disruption of FAD2-2microsomal omega-6 desaturase in soybean (Glycine max Ali Z , Aman R , Mahas A , et al. , 2020 . iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2 . Virus Res , 288 : 198129 . https://doi.org/10.1016/j.virusres.2020.198129 https://doi.org/10.1016/j.virusres.2020.198129 iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2 Altenbach SB , Chang HC , Yu XB , et al. , 2019 . Elimination of omega-1,2 gliadins from bread wheat ( Triticum aestivum ) flour: effects on immunogenic potential and end-use quality . Front Plant Sci , 10 : 580 . https://doi.org/10.3389/fpls.2019.00580 https://doi.org/10.3389/fpls.2019.00580 Elimination of omega-1,2 gliadins from bread wheat (Triticum aestivum) flour: effects on immunogenic potential and end-use quality Andersson M , Turesson H , Olsson N , et al. , 2018 . Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery . Physiol Plant , 164 ( 4 ): 378 - 384 . https://doi.org/10.1111/ppl.12731 https://doi.org/10.1111/ppl.12731 Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery Anzalone AV , Randolph PB , Davis JR , et al. , 2019 . Search-and-replace genome editing without double-strand breaks or donor DNA . Nature , 576 ( 7785 ): 149 - 157 . https://doi.org/10.1038/s41586-019-1711-4 https://doi.org/10.1038/s41586-019-1711-4 Search-and-replace genome editing without double-strand breaks or donor DNA Aravalli RN , Steer CJ , 2016 . Gene editing technology as an approach to the treatment of liver diseases . Exp Opin Biol Ther , 16 ( 5 ): 595 - 608 . https://doi.org/10.1517/14712598.2016.1158808 https://doi.org/10.1517/14712598.2016.1158808 Gene editing technology as an approach to the treatment of liver diseases Ashikari M , Sakakibara H , Lin SY , et al. , 2005 . Cytokinin oxidase regulates rice grain production . Science , 309 ( 5735 ): 741 - 745 . https://doi.org/10.1126/science.1113373 https://doi.org/10.1126/science.1113373 Cytokinin oxidase regulates rice grain production Aznar-Moreno JA , Durrett TP , 2017 . Simultaneous targeting of multiple gene homeologs to alter seed oil production in Camelina sativa . Plant Cell Physiol , 58 ( 7 ): 1260 - 1267 . https://doi.org/10.1093/pcp/pcx058 https://doi.org/10.1093/pcp/pcx058 Simultaneous targeting of multiple gene homeologs to alter seed oil production in Camelina sativa Baker M , 2012 . Gene-editing nucleases . Nat Methods , 9 ( 1 ): 23 - 26 . https://doi.org/10.1038/nmeth.1807 https://doi.org/10.1038/nmeth.1807 Gene-editing nucleases Bañuls L , Pellicer D , Castillo S , et al. , 2020 . Gene therapy in rare respiratory diseases: what have we learned so far? J Clin Med , 9 ( 8 ): 2577 . https://doi.org/10.3390/jcm9082577 https://doi.org/10.3390/jcm9082577 Gene therapy in rare respiratory diseases: what have we learned so far? Barro F , Iehisa JCM , Giménez MJ , et al. , 2016 . Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins . Plant Biotechnol J , 14 ( 3 ): 986 - 996 . https://doi.org/10.1111/pbi.12455 https://doi.org/10.1111/pbi.12455 Targeting of prolamins by RNAi in bread wheat: effectiveness of seven silencing-fragment combinations for obtaining lines devoid of coeliac disease epitopes from highly immunogenic gliadins Becker D , Wieser H , Koehler P , et al. , 2012 . Protein composition and techno-functional properties of transgenic wheat with reduced α-gliadin content obtained by RNA interference . J Appl Bot Food Qual , 85 ( 1 ): 23 - 33 . Protein composition and techno-functional properties of transgenic wheat with reduced α-gliadin content obtained by RNA interference Bogdanović O , Lister R , 2017 . DNA methylation and the preservation of cell identity . Curr Opin Genet Dev , 46 : 9 - 14 . https://doi.org/10.1016/j.gde.2017.06.007 https://doi.org/10.1016/j.gde.2017.06.007 DNA methylation and the preservation of cell identity Bouzroud S , Gasparini K , Hu GJ , et al. , 2020 . Down regulation and loss of Auxin Response Factor 4 function using CRISPR/Cas9 alters plant growth, stomatal function and improves tomato tolerance to salinity and osmotic stress . Genes , 11 ( 3 ): 272 . https://doi.org/10.3390/genes11030272 https://doi.org/10.3390/genes11030272 Down regulation and loss of Auxin ResponseFactor4 function using CRISPR/Cas9 alters plant growth, stomatal function and improves tomato tolerance to salinity and osmotic stress Braatz J , Harloff HJ , Mascher M , et al. , 2017 . CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape ( Brassica napus ) . Plant Physiol , 174 ( 2 ): 935 - 942 . https://doi.org/10.1104/pp.17.00426 https://doi.org/10.1104/pp.17.00426 CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus) Brandsma E , Verhagen HJMP , van de Laar TJW , et al. , 2021 . Rapid, sensitive, and specific severe acute respiratory syndrome coronavirus 2 detection: a multicenter comparison between standard quantitative reverse-transcriptase polymerase chain reaction and CRISPR-based DETECTR . J Infect Dis , 223 ( 2 ): 206 - 213 . https://doi.org/10.1093/infdis/jiaa641 https://doi.org/10.1093/infdis/jiaa641 Rapid, sensitive, and specific severe acute respiratory syndrome coronavirus 2 detection: a multicenter comparison between standard quantitative reverse-transcriptase polymerase chain reaction and CRISPR-based DETECTR Broughton JP , Deng XD , Yu GX , et al. , 2020 . CRISPR-Cas12-based detection of SARS-CoV-2 . Nat Biotechnol , 38 ( 7 ): 870 - 874 . https://doi.org/10.1038/s41587-020-0513-4 https://doi.org/10.1038/s41587-020-0513-4 CRISPR-Cas12-based detection of SARS-CoV-2 Bungsy M , Palmer MCL , Jeusset LM , et al. , 2021 . Reduced RBX1 expression induces chromosome instability and promotes cellular transformation in high-grade serous ovarian cancer precursor cells . Cancer Lett , 500 : 194 - 207 . https://doi.org/10.1016/j.canlet.2020.11.051 https://doi.org/10.1016/j.canlet.2020.11.051 Reduced RBX1 expression induces chromosome instability and promotes cellular transformation in high-grade serous ovarian cancer precursor cells Burstein D , Harrington LB , Strutt SC , et al. , 2017 . New CRISPR-Cas systems from uncultivated microbes . Nature , 542 ( 7640 ): 237 - 241 . https://doi.org/10.1038/nature21059 https://doi.org/10.1038/nature21059 New CRISPR-Cas systems from uncultivated microbes Büschges R , Hollricher K , Panstruga R , et al. , 1997 . The barley mlo gene: a novel control element of plant pathogen resistance . Cell , 88 ( 5 ): 695 - 705 . https://doi.org/10.1016/s0092-8674(00)81912-1 https://doi.org/10.1016/s0092-8674(00)81912-1 The barley mlo gene: a novel control element of plant pathogen resistance Cai L , Zhang L , Fu QT , et al. , 2018 . Identification and expression analysis of cytokinin metabolic genes IPTs , CYP735A and CKXs in the biofuel plant Jatropha curcas . PeerJ , 6 : e4812 . https://doi.org/10.7717/peerj.4812 https://doi.org/10.7717/peerj.4812 Identification and expression analysis of cytokinin metabolic genes IPTs, CYP735A and CKXs in the biofuel plant Jatropha curcas Cao XS , Kouyama-Suzuki E , Pang B , et al. , 2020 . Inhibition of DNA ligase IV enhances the CRISPR/Cas9-mediated knock-in efficiency in mouse brain neurons . Biochem Biophys Res Commun , 533 ( 3 ): 449 - 457 . https://doi.org/10.1016/j.bbrc.2020.09.053 https://doi.org/10.1016/j.bbrc.2020.09.053 Inhibition of DNA ligase IV enhances the CRISPR/Cas9-mediated knock-in efficiency in mouse brain neurons Čermák T , Baltes NJ , Čegan R , et al. , 2015 . High-frequency, precise modification of the tomato genome . Genome Biol , 16 : 232 . https://doi.org/10.1186/s13059-015-0796-9 https://doi.org/10.1186/s13059-015-0796-9 High-frequency, precise modification of the tomato genome Chandrasekaran J , Brumin M , Wolf D , et al. , 2016 . Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology . Mol Plant Pathol , 17 ( 7 ): 1140 - 1153 . https://doi.org/10.1111/mpp.12375 https://doi.org/10.1111/mpp.12375 Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology Charrier A , Vergne E , Dousset N , et al. , 2019 . Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR-Cas9 system . Front Plant Sci , 10 : 40 . https://doi.org/10.3389/fpls.2019.00040 https://doi.org/10.3389/fpls.2019.00040 Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR-Cas9 system Chatterjee P , Jakimo N , Jacobson JM , 2018 . Minimal PAM specificity of a highly similar SpCas9 ortholog . Sci Adv , 4 ( 10 ): eaau0766 . https://doi.org/10.1126/sciadv.aau0766 https://doi.org/10.1126/sciadv.aau0766 Minimal PAM specificity of a highly similar SpCas9 ortholog Chavez A , Scheiman J , Vora S , et al. , 2015 . Highly efficient Cas9-mediated transcriptional programming . Nat Methods , 12 ( 4 ): 326 - 328 . https://doi.org/10.1038/nmeth.3312 https://doi.org/10.1038/nmeth.3312 Highly efficient Cas9-mediated transcriptional programming Chen C , Liu Y , Rappaport AR , et al. , 2014 . MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia . Cancer Cell , 25 ( 5 ): 652 - 665 . https://doi.org/10.1016/j.ccr.2014.03.016 https://doi.org/10.1016/j.ccr.2014.03.016 MLL3 is a haploinsufficient 7q tumor suppressor in acute myeloid leukemia Chen HR , Gao SS , Liu WX , et al. , 2021 . RNA N 6 -methyladenosine methyltransferase METTL3 facilitates colorectal cancer by activating the m 6 A-GLUT1-mTORC1 axis and is a therapeutic target . Gastroenterology , 160 ( 4 ): 1284 - 1300.e16 . https://doi.org/10.1053/j.gastro.2020.11.013 https://doi.org/10.1053/j.gastro.2020.11.013 RNA N6-methyladenosine methyltransferase METTL3 facilitates colorectal cancer by activating the m6A-GLUT1-mTORC1 axis and is a therapeutic target Chen JS , Ma EB , Harrington LB , et al. , 2018 . CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNAse activity . Science , 360 ( 6387 ): 436 - 439 . https://doi.org/10.1126/science.aar6245 https://doi.org/10.1126/science.aar6245 CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNAse activity Chen KL , Wang YP , Zhang R , et al. , 2019 . CRISPR/Cas genome editing and precision plant breeding in agriculture . Annu Rev Plant Biol , 70 : 667 - 697 . https://doi.org/10.1146/annurev-arplant-050718-100049 https://doi.org/10.1146/annurev-arplant-050718-100049 CRISPR/Cas genome editing and precision plant breeding in agriculture Chen LY , Nan HY , Kong LP , et al. , 2020 . Soybean AP1 homologs control flowering time and plant height . J Integr Plant Biol , 62 ( 12 ): 1868 - 1879 . https://doi.org/10.1111/jipb.12988 https://doi.org/10.1111/jipb.12988 Soybean AP1 homologs control flowering time and plant height Chen YZ , Fu MC , Li H , et al. , 2021 . High-oleic acid content, nontransgenic allotetraploid cotton ( Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system . Plant Biotechnol J , 19 ( 3 ): 424 - 426 . https://doi.org/10.1111/pbi.13507 https://doi.org/10.1111/pbi.13507 High-oleic acid content, nontransgenic allotetraploid cotton (Gossypium hirsutum L.) generated by knockout of GhFAD2 genes with CRISPR/Cas9 system Cheng AW , Wang HY , Yang H , et al. , 2013 . Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system . Cell Res , 23 ( 10 ): 1163 - 1171 . https://doi.org/10.1038/cr.2013.122 https://doi.org/10.1038/cr.2013.122 Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system Cheng Q , Dong LD , Su T , et al. , 2019 . CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean . BMC Plant Biol , 19 : 562 . https://doi.org/10.1186/s12870-019-2145-8 https://doi.org/10.1186/s12870-019-2145-8 CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean Chertow DS , 2018 . Next-generation diagnostics with CRISPR . Science , 360 ( 6387 ): 381 - 382 . https://doi.org/10.1126/science.aat4982 https://doi.org/10.1126/science.aat4982 Next-generation diagnostics with CRISPR Choi BD , Yu XL , Castano AP , et al. , 2019 . CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma . J Immunother Cancer , 7 ( 1 ): 304 . https://doi.org/10.1186/s40425-019-0806-7 https://doi.org/10.1186/s40425-019-0806-7 CRISPR-Cas9 disruption of PD-1 enhances activity of universal EGFRvIII CAR T cells in a preclinical model of human glioblastoma Correa-Aragunde N , Graziano M , Lamattina L , 2004 . Nitric oxide plays a central role in determining lateral root development in tomato . Planta , 218 ( 6 ): 900 - 905 . https://doi.org/10.1007/s00425-003-1172-7 https://doi.org/10.1007/s00425-003-1172-7 Nitric oxide plays a central role in determining lateral root development in tomato Cui Y , Jiang N , Xu ZJ , et al. , 2020 . Heterotrimeric G protein are involved in the regulation of multiple agronomic traits and stress tolerance in rice . BMC Plant Biol , 20 : 90 . https://doi.org/10.1186/s12870-020-2289-6 https://doi.org/10.1186/s12870-020-2289-6 Heterotrimeric G protein are involved in the regulation of multiple agronomic traits and stress tolerance in rice Dar AA , Choudhury AR , Kancharla PK , et al. , 2017 . The FAD2 gene in plants: occurrence, regulation, and role . Front Plant Sci , 8 : 1789 . https://doi.org/10.3389/fpls.2017.01789 https://doi.org/10.3389/fpls.2017.01789 The FAD2 gene in plants: occurrence, regulation, and role Deng H , Tan SW , Gao XQ , et al. , 2020 . Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity . Acta Pharm Sin B , 10 ( 2 ): 358 - 373 . https://doi.org/10.1016/j.apsb.2019.07.004 https://doi.org/10.1016/j.apsb.2019.07.004 Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity Dickinson DJ , Ward JD , Reiner DJ , et al. , 2013 . Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination . Nat Methods , 10 ( 10 ): 1028 - 1034 . https://doi.org/10.1038/nmeth.2641 https://doi.org/10.1038/nmeth.2641 Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination Ding X , Yin K , Li ZY , et al. , 2020 . Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay . Nat Commun , 11 : 4711 . https://doi.org/10.1038/s41467-020-18575-6 https://doi.org/10.1038/s41467-020-18575-6 Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay Doman JL , Raguram A , Newby GA , et al. , 2020 . Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors . Nat Biotechnol , 38 ( 5 ): 620 - 628 . https://doi.org/10.1038/s41587-020-0414-6 https://doi.org/10.1038/s41587-020-0414-6 Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors Doudna JA , 2020 . The promise and challenge of therapeutic genome editing . Nature , 578 ( 7794 ): 229 - 236 . https://doi.org/10.1038/s41586-020-1978-5 https://doi.org/10.1038/s41586-020-1978-5 The promise and challenge of therapeutic genome editing Du J , Duan S , Wang H , et al. , 2008 . Comprehensive analysis of polymorphisms throughout GAD1 gene: a family-based association study in schizophrenia . J Neural Transm (Vienna) , 115 ( 3 ): 513 - 519 . https://doi.org/10.1007/s00702-007-0844-z https://doi.org/10.1007/s00702-007-0844-z Comprehensive analysis of polymorphisms throughout GAD1 gene: a family-based association study in schizophrenia Ellis BL , Hirsch ML , Porter SN , et al. , 2013 . Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by food and drug administration-approved drugs . Gene Ther , 20 ( 1 ): 35 - 42 . https://doi.org/10.1038/gt.2011.211 https://doi.org/10.1038/gt.2011.211 Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by food and drug administration-approved drugs Endo M , Mikami M , Endo A , et al. , 2019 . Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM . Nat Plants , 5 ( 1 ): 14 - 17 . https://doi.org/10.1038/s41477-018-0321-8 https://doi.org/10.1038/s41477-018-0321-8 Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM Esvelt KM , Mali P , Braff JL , et al. , 2013 . Orthogonal Cas9 proteins for RNA-guided gene regulation and editing . Nature Methods , 10 ( 11 ): 1116 - 1121 . https://doi.org/10.1038/Nmeth.2681 https://doi.org/10.1038/Nmeth.2681 Orthogonal Cas9 proteins for RNA-guided gene regulation and editing Feng YL , Liu SC , Chen RD , et al. , 2021 . Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 22 ( 1 ): 73 - 86 . https://doi.org/10.1631/jzus.B2000282 https://doi.org/10.1631/jzus.B2000282 Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing Ferrari G , Thrasher AJ , Aiuti A , 2021 . Gene therapy using haematopoietic stem and progenitor cells . Nat Rev Genet , 22 : 216 - 234 . https://doi.org/10.1038/s41576-020-00298-5 https://doi.org/10.1038/s41576-020-00298-5 Gene therapy using haematopoietic stem and progenitor cells Filiz E , Vatansever R , 2018 . Genome-wide identification of mildew resistance locus O ( MLO ) genes in tree model poplar ( Populus trichocarpa ): powdery mildew management in woody plants . Eur J Plant Pathol , 152 ( 1 ): 95 - 109 . https://doi.org/10.1007/s10658-018-1454-3 https://doi.org/10.1007/s10658-018-1454-3 Genome-wide identification of mildew resistance locus O (MLO) genes in tree model poplar (Populus trichocarpa): powdery mildew management in woody plants Flores T , Todd CD , Tovar-Mendez A , et al. , 2008 . Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development . Plant Physiol , 147 ( 4 ): 1936 - 1946 . https://doi.org/10.1104/pp.108.121459 https://doi.org/10.1104/pp.108.121459 Arginase-negative mutants of Arabidopsis exhibit increased nitric oxide signaling in root development Fu Y , Rocha PP , Luo VM , et al. , 2016 . CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci . Nat Commun , 7 : 11707 . https://doi.org/10.1038/ncomms11707 https://doi.org/10.1038/ncomms11707 CRISPR-dCas9 and sgRNA scaffolds enable dual-colour live imaging of satellite sequences and repeat-enriched individual loci Fujihara K , Yamada K , Ichitani Y , et al. , 2020 . CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia . Transl Psychiatry , 10 : 426 . https://doi.org/10.1038/s41398-020-01108-6 https://doi.org/10.1038/s41398-020-01108-6 CRISPR/Cas9-engineered Gad1 elimination in rats leads to complex behavioral changes: implications for schizophrenia Furukawa T , Maekawa M , Oki T , et al. , 2007 . The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp . Plant J , 49 ( 1 ): 91 - 102 . https://doi.org/10.1111/j.1365-313X.2006.02958.x https://doi.org/10.1111/j.1365-313X.2006.02958.x The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp Gao LY , Cox DBT , Yan WX , et al. , 2017 . Engineered Cpf1 variants with altered PAM specificities . Nat Biotechnol , 35 ( 8 ): 789 - 792 . https://doi.org/10.1038/nbt.3900 https://doi.org/10.1038/nbt.3900 Engineered Cpf1 variants with altered PAM specificities Gao SP , Kiliti AJ , Zhang K , et al. , 2021 . AKT1 E17K inhibits cancer cell migration by abrogating β-catenin signaling . Mol Cancer Res , online . https://doi.org/10.1158/1541-7786.Mcr-20-0623 https://doi.org/10.1158/1541-7786.Mcr-20-0623 AKT1 E17K inhibits cancer cell migration by abrogating β-catenin signaling Garneau JE , Dupuis MÈ , Villion M , et al. , 2010 . The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA . Nature , 468 ( 7320 ): 67 - 71 . https://doi.org/10.1038/nature09523 https://doi.org/10.1038/nature09523 The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Gasiunas G , Barrangou R , Horvath P , et al. , 2012 . Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria . Proc Natl Acad Sci USA , 109 ( 39 ): E2579 - E2586 . Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria Gaudelli NM , Komor AC , Rees HA , et al. , 2017 . Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage . Nature , 551 ( 7681 ): 464 - 471 . https://doi.org/10.1038/nature24644 https://doi.org/10.1038/nature24644 Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage Gaudelli NM , Lam DK , Rees HA , et al. , 2020 . Directed evolution of adenine base editors with increased activity and therapeutic application . Nat Biotechnol , 38 ( 7 ): 892 - 900 . https://doi.org/10.1038/s41587-020-0491-6 https://doi.org/10.1038/s41587-020-0491-6 Directed evolution of adenine base editors with increased activity and therapeutic application Ge ZX , Zheng LQ , Zhao YL , et al. , 2019 . Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants . Plant Biotechnol J , 17 ( 10 ): 1865 - 1867 . https://doi.org/10.1111/pbi.13148 https://doi.org/10.1111/pbi.13148 Engineered xCas9 and SpCas9-NG variants broaden PAM recognition sites to generate mutations in Arabidopsis plants Gebert LFR , MacRae IJ , 2019 . Regulation of microRNA function in animals . Nat Rev Mol Cell Biol , 20 ( 1 ): 21 - 37 . https://doi.org/10.1038/s41580-018-0045-7 https://doi.org/10.1038/s41580-018-0045-7 Regulation of microRNA function in animals Gehrke JM , Cervantes O , Clement MK , et al. , 2018 . An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities . Nat Biotechnol , 36 ( 10 ): 977 - 982 . https://doi.org/10.1038/nbt.4199 https://doi.org/10.1038/nbt.4199 An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities Gilbert LA , Larson MH , Morsut L , et al. , 2013 . CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes . Cell , 154 ( 2 ): 442 - 451 . https://doi.org/10.1016/j.cell.2013.06.044 https://doi.org/10.1016/j.cell.2013.06.044 CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes Gilbert LA , Horlbeck MA , Adamson B , et al. , 2014 . Genome-scale CRISPR-mediated control of gene repression and activation . Cell , 159 ( 3 ): 647 - 661 . https://doi.org/10.1016/j.cell.2014.09.029 https://doi.org/10.1016/j.cell.2014.09.029 Genome-scale CRISPR-mediated control of gene repression and activation Gil-Humanes J , Pistón F , Hernando A , et al. , 2008 . Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat . J Cereal Sci , 48 ( 3 ): 565 - 568 . https://doi.org/10.1016/j.jcs.2008.03.005 https://doi.org/10.1016/j.jcs.2008.03.005 Silencing of γ-gliadins by RNA interference (RNAi) in bread wheat Gil-Humanes J , Pistón F , Tollefsen S , et al. , 2010 . Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference . Proc Natl Acad Sci USA , 107 ( 39 ): 17023 - 17028 . https://doi.org/10.1073/pnas.1007773107 https://doi.org/10.1073/pnas.1007773107 Effective shutdown in the expression of celiac disease-related wheat gliadin T-cell epitopes by RNA interference Gleditzsch D , Pausch P , Müller-Esparza H , et al. , 2019 . PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures . RNA Biol , 16 ( 4 ): 504 - 517 . https://doi.org/10.1080/15476286.2018.1504546 https://doi.org/10.1080/15476286.2018.1504546 PAM identification by CRISPR-Cas effector complexes: diversified mechanisms and structures González MN , Massa GA , Andersson M , et al. , 2020 . Reduced enzymatic browning in potato tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein complexes delivery of the CRISPR/Cas9 system . Front Plant Sci , 10 : 1649 . https://doi.org/10.3389/fpls.2019.01649 https://doi.org/10.3389/fpls.2019.01649 Reduced enzymatic browning in potato tubers by specific editing of a polyphenol oxidase gene via ribonucleoprotein complexes delivery of the CRISPR/Cas9 system Gootenberg JS , Abudayyeh OO , Lee JW , et al. , 2017 . Nucleic acid detection with CRISPR-Cas13a/C2c2 . Science , 356 ( 6336 ): 438 - 442 . https://doi.org/10.1126/science.aam9321 https://doi.org/10.1126/science.aam9321 Nucleic acid detection with CRISPR-Cas13a/C2c2 Gootenberg JS , Abudayyeh OO , Kellner MJ , et al. , 2018 . Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 . Science , 360 ( 6387 ): 439 - 444 . https://doi.org/10.1126/science.aaq0179 https://doi.org/10.1126/science.aaq0179 Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 Haas KT , Wightman R , Meyerowitz EM , et al. , 2020 . Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells . Science , 367 ( 6481 ): 1003 - 1007 . https://doi.org/10.1126/science.aaz5103 https://doi.org/10.1126/science.aaz5103 Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells Han LQ , Zhang ML , Xing ZY , et al. , 2020 . Knockout of butyrophilin subfamily 1 member A1 ( BTN1A1 ) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells . J Anim Sci Biotechnol , 11 : 72 . https://doi.org/10.1186/s40104-020-00479-6 https://doi.org/10.1186/s40104-020-00479-6 Knockout of butyrophilin subfamily 1 member A1 (BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells Hanzawa N , Hashimoto K , Yuan XM , et al. , 2020 . Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing . Sci Rep , 10 : 5181 . https://doi.org/10.1038/s41598-020-62035-6 https://doi.org/10.1038/s41598-020-62035-6 Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing Hayward D , Cole PA , 2016 . LSD1 histone demethylase assays and inhibition . Methods Enzymol , 573 : 261 - 278 . https://doi.org/10.1016/bs.mie.2016.01.020 https://doi.org/10.1016/bs.mie.2016.01.020 LSD1 histone demethylase assays and inhibition He SH , 2020 . The first human trial of CRISPR-based cell therapy clears safety concerns as new treatment for late-stage lung cancer . Signal Transduct Target Ther , 5 : 168 . https://doi.org/10.1038/s41392-020-00283-8 https://doi.org/10.1038/s41392-020-00283-8 The first human trial of CRISPR-based cell therapy clears safety concerns as new treatment for late-stage lung cancer Hermans PW , van Soolingen D , Bik EM , et al. , 1991 . Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains . Infect Immun , 59 ( 8 ): 2695 - 2705 . https://doi.org/10.1128/IAI.59.8.2695-2705.1991 https://doi.org/10.1128/IAI.59.8.2695-2705.1991 Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains Heyer WD , Ehmsen KT , Liu J , 2010 . Regulation of homologous recombination in eukaryotes . Annu Rev Genet , 44 : 113 - 139 . https://doi.org/10.1146/annurev-genet-051710-150955 https://doi.org/10.1146/annurev-genet-051710-150955 Regulation of homologous recombination in eukaryotes Hirano S , Nishimasu H , Ishitani R , et al. , 2016 . Structural basis for the altered PAM specificities of engineered CRISPR-Cas9 . Mol Cell , 61 ( 6 ): 886 - 894 . https://doi.org/10.1016/j.molcel.2016.02.018 https://doi.org/10.1016/j.molcel.2016.02.018 Structural basis for the altered PAM specificities of engineered CRISPR-Cas9 Honma Y , Adhikari PB , Kuwata K , et al. , 2020 . High-quality sugar production by osgcs1 rice . Commun Biol , 3 : 617 . https://doi.org/10.1038/s42003-020-01329-x https://doi.org/10.1038/s42003-020-01329-x High-quality sugar production by osgcs1 rice Horii T , Morita S , Hino S , et al. , 2020 . Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome . Genome Biol , 21 : 77 . https://doi.org/10.1186/s13059-020-01991-8 https://doi.org/10.1186/s13059-020-01991-8 Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome Hou ZG , Zhang Y , Propson NE , et al. , 2013 . Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis . Proc Natl Acad Sci USA , 110 ( 39 ): 15644 - 15649 . https://doi.org/10.1073/pnas.1313587110 https://doi.org/10.1073/pnas.1313587110 Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis Hu JH , Miller SM , Geurts MH , et al. , 2018 . Evolved Cas9 variants with broad PAM compatibility and high DNA specificity . Nature , 556 ( 7699 ): 57 - 63 . https://doi.org/10.1038/nature26155 https://doi.org/10.1038/nature26155 Evolved Cas9 variants with broad PAM compatibility and high DNA specificity Hu XX , Wang C , Fu YP , et al. , 2016 . Expanding the range of CRISPR/Cas9 genome editing in rice . Mol Plant , 9 ( 6 ): 943 - 945 . https://doi.org/10.1016/j.molp.2016.03.003 https://doi.org/10.1016/j.molp.2016.03.003 Expanding the range of CRISPR/Cas9 genome editing in rice Hu XX , Meng XB , Liu Q , et al. , 2018 . Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice . Plant Biotechnol J , 16 ( 1 ): 292 - 297 . https://doi.org/10.1111/pbi.12771 https://doi.org/10.1111/pbi.12771 Increasing the efficiency of CRISPR-Cas9-VQR precise genome editing in rice Hua K , Tao XP , Liang WY , et al. , 2020 . Simplified adenine base editors improve adenine base editing efficiency in rice . Plant Biotechnol J , 18 ( 3 ): 770 - 778 . https://doi.org/10.1111/pbi.13244 https://doi.org/10.1111/pbi.13244 Simplified adenine base editors improve adenine base editing efficiency in rice Huang H , Liu RE , Niu QF , et al. , 2019 . Global increase in DNA methylation during orange fruit development and ripening . Proc Natl Acad Sci USA , 116 ( 4 ): 1430 - 1436 . https://doi.org/10.1073/pnas.1815441116 https://doi.org/10.1073/pnas.1815441116 Global increase in DNA methylation during orange fruit development and ripening Huang HB , Cui TT , Zhang LL , et al. , 2020 . Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus . Theor Appl Genet , 133 ( 8 ): 2401 - 2411 . https://doi.org/10.1007/s00122-020-03607-y https://doi.org/10.1007/s00122-020-03607-y Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus Hunziker J , Nishida K , Kondo A , et al. , 2020 . Multiple gene substitution by target-AID base-editing technology in tomato . Sci Rep , 10 : 20471 . https://doi.org/10.1038/s41598-020-77379-2 https://doi.org/10.1038/s41598-020-77379-2 Multiple gene substitution by target-AID base-editing technology in tomato Huo WY , Zhao GN , Yin JG , et al. , 2017 . Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells . J Cancer , 8 ( 1 ): 57 - 64 . https://doi.org/10.7150/jca.16723 https://doi.org/10.7150/jca.16723 Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells Ishino Y , Shinagawa H , Makino K , et al. , 1987 . Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli , and identification of the gene product . J Bacteriol , 169 ( 12 ): 5429 - 5433 . https://doi.org/10.1128/jb.169.12.5429-5433.1987 https://doi.org/10.1128/jb.169.12.5429-5433.1987 Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product Islam KU , Iqbal J , 2020 . An update on molecular diagnostics for COVID-19 . Front Cell Infect Microbiol , 10 : 560616 . https://doi.org/10.3389/fcimb.2020.560616 https://doi.org/10.3389/fcimb.2020.560616 An update on molecular diagnostics for COVID-19 Ito Y , Nishizawa-Yokoi A , Endo M , et al. , 2015 . CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening . Biochem Biophys Res Commun , 467 ( 1 ): 76 - 82 . https://doi.org/10.1016/j.bbrc.2015.09.117 https://doi.org/10.1016/j.bbrc.2015.09.117 CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening Jacobs TB , Lafayette PR , Schmitz RJ , et al. , 2015 . Targeted genome modifications in soybean with CRISPR/Cas9 . BMC Biotechnol , 15 : 16 . https://doi.org/10.1186/s12896-015-0131-2 https://doi.org/10.1186/s12896-015-0131-2 Targeted genome modifications in soybean with CRISPR/Cas9 Jansen R , van Embden JDA , Gaastra W , et al. , 2002 . Identification of genes that are associated with DNA repeats in prokaryotes . Mol Microbiol , 43 ( 6 ): 1565 - 1575 . https://doi.org/10.1046/j.1365-2958.2002.02839.x https://doi.org/10.1046/j.1365-2958.2002.02839.x Identification of genes that are associated with DNA repeats in prokaryotes Javalkote VS , Kancharla N , Bhadra B , et al. , 2020 . CRISPR-based assays for rapid detection of SARS-CoV-2 . Methods , in press . https://doi.org/10.1016/j.ymeth.2020.10.003 https://doi.org/10.1016/j.ymeth.2020.10.003 CRISPR-based assays for rapid detection of SARS-CoV-2 Jia HG , Wang N , 2014 . Targeted genome editing of sweet orange using Cas9/sgRNA . PLoS ONE , 9 ( 4 ): e93806 . https://doi.org/10.1371/journal.pone.0093806 https://doi.org/10.1371/journal.pone.0093806 Targeted genome editing of sweet orange using Cas9/sgRNA Jiang FN , Liang YX , Wei W , et al. , 2020 . Functional classification of prostate cancer-associated miRNAs through CRISPR/Cas9-mediated gene knockout . Mol Med Rep , 22 ( 5 ): 3777 - 3784 . https://doi.org/10.3892/mmr.2020.11491 https://doi.org/10.3892/mmr.2020.11491 Functional classification of prostate cancer-associated miRNAs through CRISPR/Cas9-mediated gene knockout Jiang WZ , Zhou HB , Bi HH , et al. , 2013 . Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice . Nucleic Acids Res , 41 ( 20 ): e188 . https://doi.org/10.1093/nar/gkt780 https://doi.org/10.1093/nar/gkt780 Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice Jiang WZ , Henry IM , Lynagh PG , et al. , 2017 . Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa , using CRISPR/Cas9 gene editing . Plant Biotechnol J , 15 ( 5 ): 648 - 657 . https://doi.org/10.1111/pbi.12663 https://doi.org/10.1111/pbi.12663 Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing Jin Y , Datsomor AK , Olsen RE , et al. , 2020 . Targeted mutagenesis of ∆5 and ∆6 fatty acyl desaturases induce dysregulation of lipid metabolism in Atlantic salmon ( Salmo salar ) . BMC Genomics , 21 : 805 . https://doi.org/10.1186/s12864-020-07218-1 https://doi.org/10.1186/s12864-020-07218-1 Targeted mutagenesis of ∆5 and ∆6 fatty acyl desaturases induce dysregulation of lipid metabolism in Atlantic salmon (Salmo salar) Jinek M , Chylinski K , Fonfara I , et al. , 2012 . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity . Science , 337 ( 6096 ): 816 - 821 . https://doi.org/10.1126/science.1225829 https://doi.org/10.1126/science.1225829 A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity Jouanin A , Schaart JG , Boyd LA , et al. , 2019 . Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families . BMC Plant Biol , 19 : 333 . https://doi.org/10.1186/s12870-019-1889-5 https://doi.org/10.1186/s12870-019-1889-5 Outlook for coeliac disease patients: towards bread wheat with hypoimmunogenic gluten by gene editing of α- and γ-gliadin gene families Joung J , Engreitz JM , Konermann S , et al. , 2017 . Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood . Nature , 548 ( 7667 ): 343 - 346 . https://doi.org/10.1038/nature23451 https://doi.org/10.1038/nature23451 Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood Juhász A , Colgrave ML , Howitt CA , 2020 . Developing gluten-free cereals and the role of proteomics in product safety . J Cereal Sci , 93 : 102932 . https://doi.org/10.1016/j.jcs.2020.102932 https://doi.org/10.1016/j.jcs.2020.102932 Developing gluten-free cereals and the role of proteomics in product safety Jung YJ , Lee GJ , Bae S , et al. , 2018 . Reduced ethylene production in tomato fruits upon CRSPR/Cas9-mediated leMADS-RIN mutagenesis . Hortic Sci Technol , 36 ( 3 ): 396 - 405 . https://doi.org/10.12972/kjhst.20180039 https://doi.org/10.12972/kjhst.20180039 Reduced ethylene production in tomato fruits upon CRSPR/Cas9-mediated leMADS-RIN mutagenesis Kang BC , Yun JY , Kim ST , et al. , 2018 . Precision genome engineering through adenine base editing in plants . Nat Plants , 4 : 427 - 431 . https://doi.org/10.1038/s41477-018-0178-x https://doi.org/10.1038/s41477-018-0178-x Precision genome engineering through adenine base editing in plants Kanitchinda S , Srisala J , Suebsing R , et al. , 2020 . CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei . Biotechnol Rep (Amst) , 27 : e00485 . https://doi.org/10.1016/j.btre.2020.e00485 https://doi.org/10.1016/j.btre.2020.e00485 CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei Karunarathna NL , Wang HY , Harloff HJ , et al. , 2020 . Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes . Plant Biotechnol J , 18 ( 11 ): 2251 - 2266 . https://doi.org/10.1111/pbi.13381 https://doi.org/10.1111/pbi.13381 Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACIDREDUCER genes Kaur N , Alok A , Shivani , et al. , 2020 . CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit . Metab Eng , 59 : 76 - 86 . https://doi.org/10.1016/j.ymben.2020.01.008 https://doi.org/10.1016/j.ymben.2020.01.008 CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit Kaya H , Mikami M , Endo A , et al. , 2016 . Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9 . Sci Rep , 6 : 26871 . https://doi.org/10.1038/Srep26871 https://doi.org/10.1038/Srep26871 Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9 Kearns NA , Pham H , Tabak B , et al. , 2015 . Functional annotation of native enhancers with a Cas9-histone demethylase fusion . Nat Methods , 12 ( 5 ): 401 - 403 . https://doi.org/10.1038/nmeth.3325 https://doi.org/10.1038/nmeth.3325 Functional annotation of native enhancers with a Cas9-histone demethylase fusion Kellner MJ , Koob JG , Gootenberg JS , et al. , 2019 . SHERLOCK: nucleic acid detection with CRISPR nucleases . Nat Protoc , 14 ( 10 ): 2986 - 3012 . https://doi.org/10.1038/s41596-019-0210-2 https://doi.org/10.1038/s41596-019-0210-2 SHERLOCK: nucleic acid detection with CRISPR nucleases Khosravi S , Ishii T , Dreissig S , et al. , 2020 . Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells . Chromosome Res , 28 ( 1 ): 7 - 17 . https://doi.org/10.1007/s10577-019-09622-0 https://doi.org/10.1007/s10577-019-09622-0 Application and prospects of CRISPR/Cas9-based methods to trace defined genomic sequences in living and fixed plant cells Kim E , Koo T , Park SW , et al. , 2017 . In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni . Nat Commun , 8 : 14500 . https://doi.org/10.1038/Ncomms14500 https://doi.org/10.1038/Ncomms14500 In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni Kim H , Kim ST , Ryu J , et al. , 2017 . CRISPR/Cpf1-mediated DNA-free plant genome editing . Nat Commun , 8 : 14406 . https://doi.org/10.1038/Ncomms14406 https://doi.org/10.1038/Ncomms14406 CRISPR/Cpf1-mediated DNA-free plant genome editing Kim YA , Moon H , Park CJ , 2019 . CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv . oryzae. Rice , 12 : 67 . https://doi.org/10.1186/s12284-019-0325-7 https://doi.org/10.1186/s12284-019-0325-7 CRISPR/Cas9-targeted mutagenesis of Os8N3 in rice to confer resistance to Xanthomonas oryzae pv Kim YB , Komor AC , Levy JM , et al. , 2017 . Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions . Nat Biotechnol , 35 ( 4 ): 371 - 376 . https://doi.org/10.1038/nbt.3803 https://doi.org/10.1038/nbt.3803 Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions Kitamoto K , Taketani Y , Fujii W , et al. , 2020 . Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair . Sci Rep , 10 : 2000 . https://doi.org/10.1038/s41598-020-58876-w https://doi.org/10.1038/s41598-020-58876-w Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair Kleinstiver BP , Prew MS , Tsai SQ , et al. , 2015a . Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition . Nat Biotechnol , 33 ( 12 ): 1293 - 1298 . https://doi.org/10.1038/nbt.3404 https://doi.org/10.1038/nbt.3404 Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition Kleinstiver BP , Prew MS , Tsai SQ , et al. , 2015b . Engineered CRISPR-Cas9 nucleases with altered PAM specificities . Nature , 523 ( 7561 ): 481 - 485 . https://doi.org/10.1038/nature14592 https://doi.org/10.1038/nature14592 Engineered CRISPR-Cas9 nucleases with altered PAM specificities Komor AC , Kim YB , Packer MS , et al. , 2016 . Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage . Nature , 533 ( 7603 ): 420 - 424 . https://doi.org/10.1038/nature17946 https://doi.org/10.1038/nature17946 Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage Komor AC , Zhao KT , Packer MS , et al. , 2017 . Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity . Sci Adv , 3 ( 8 ): eaao4774 . https://doi.org/10.1126/sciadv.aao4774 https://doi.org/10.1126/sciadv.aao4774 Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity Konermann S , Brigham MD , Trevino AE , et al. , 2013 . Optical control of mammalian endogenous transcription and epigenetic states . Nature , 500 ( 7463 ): 472 - 476 . https://doi.org/10.1038/nature12466 https://doi.org/10.1038/nature12466 Optical control of mammalian endogenous transcription and epigenetic states Konermann S , Brigham MD , Trevino AE , et al. , 2015 . Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex . Nature , 517 ( 7536 ): 583 - 588 . https://doi.org/10.1038/nature14136 https://doi.org/10.1038/nature14136 Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex Kumar P , Malik YS , Ganesh B , et al. , 2020 . CRISPR-Cas system: an approach with potentials for COVID-19 diagnosis and therapeutics . Front Cell Infect Microbiol , 10 : 576875 . https://doi.org/10.3389/fcimb.2020.576875 https://doi.org/10.3389/fcimb.2020.576875 CRISPR-Cas system: an approach with potentials for COVID-19 diagnosis and therapeutics Kurihara T , Kouyama-Suzuki E , Satoga M , et al. , 2020 . DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons . Biochem Biophys Res Commun , 524 ( 3 ): 621 - 628 . https://doi.org/10.1016/j.bbrc.2020.01.132 https://doi.org/10.1016/j.bbrc.2020.01.132 DNA repair protein RAD51 enhances the CRISPR/Cas9-mediated knock-in efficiency in brain neurons Kurt IC , Zhou RH , Iyer S , et al. , 2020 . CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells . Nat Biotechnol , 39 ( 1 ): 41 - 46 . https://doi.org/10.1038/s41587-020-0609-x https://doi.org/10.1038/s41587-020-0609-x CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells Kusano H , Ohnuma M , Mutsuro-Aoki H , et al. , 2018 . Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato . Sci Rep , 8 : 13753 . https://doi.org/10.1038/s41598-018-32049-2 https://doi.org/10.1038/s41598-018-32049-2 Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato Lau CH , Suh Y , 2018 . In vivo epigenome editing and transcriptional modulation using CRISPR technology . Transgenic Res , 27 ( 6 ): 489 - 509 . https://doi.org/10.1007/s11248-018-0096-8 https://doi.org/10.1007/s11248-018-0096-8 In vivo epigenome editing and transcriptional modulation using CRISPR technology Lawhorn IEB , Ferreira JP , Wang CL , 2014 . Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53 . PLoS ONE , 9 ( 11 ): e113232 . https://doi.org/10.1371/journal.pone.0113232 https://doi.org/10.1371/journal.pone.0113232 Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53 Li C , Unver T , Zhang BH , 2017 . A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton ( Gossypium hirsutum L.) . Sci Rep , 7 : 43902 . https://doi.org/10.1038/srep43902 https://doi.org/10.1038/srep43902 A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in cotton (Gossypium hirsutum L.) Li C , Zhang R , Meng XB , et al. , 2020 . Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors . Nat Biotechnol , 38 ( 7 ): 875 - 882 . https://doi.org/10.1038/s41587-019-0393-7 https://doi.org/10.1038/s41587-019-0393-7 Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors Li HY , Li JY , Chen JL , et al. , 2020 . Precise modifications of both exogenous and endogenous genes in rice by prime editing . Mol Plant , 13 ( 5 ): 671 - 674 . https://doi.org/10.1016/j.molp.2020.03.011 https://doi.org/10.1016/j.molp.2020.03.011 Precise modifications of both exogenous and endogenous genes in rice by prime editing Li JF , Norville JE , Aach J , et al. , 2013 . Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 . Nat Biotechnol , 31 ( 8 ): 688 - 691 . https://doi.org/10.1038/nbt.2654 https://doi.org/10.1038/nbt.2654 Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotianabenthamiana using guide RNA and Cas9 Li JY , Jiao GA , Sun YW , et al. , 2020 . Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas 9 . Plant Biotechnol J , online . https://doi.org/10.1111/pbi.13519 https://doi.org/10.1111/pbi.13519 Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas Li M , Chen R , Jiang QY , et al. , 2021 . GmNAC06 , a NAC domain transcription factor enhances salt stress tolerance in soybean . Plant Mol Biol , 105 ( 3 ): 333 - 345 . https://doi.org/10.1007/s11103-020-01091-y https://doi.org/10.1007/s11103-020-01091-y GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean Li MR , Li XX , Zhou ZJ , et al. , 2016 . Reassessment of the four yield-related genes Gn1a , DEP1 , GS3 , and IPA1 in rice using a CRISPR/Cas9 system . Front Plant Sci , 7 : 377 . https://doi.org/10.3389/fpls.2016.00377 https://doi.org/10.3389/fpls.2016.00377 Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system Li R , Fu DQ , Zhu BZ , et al. , 2018a . CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters tomato fruit ripening . Plant J , 94 ( 3 ): 513 - 524 . https://doi.org/10.1111/tpj.13872 https://doi.org/10.1111/tpj.13872 CRISPR/Cas9-mediated mutagenesis of lncRNA1459 alters tomato fruit ripening Li R , Li R , Li XD , et al. , 2018b . Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum . Plant Biotechnol J , 16 ( 2 ): 415 - 427 . https://doi.org/10.1111/pbi.12781 https://doi.org/10.1111/pbi.12781 Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum Li SB , Xie ZZ , Hu CG , et al. , 2016 . A review of auxin response factors (ARFs) in plants . Front Plant Sci , 7 : 47 . https://doi.org/10.3389/fpls.2016.00047 https://doi.org/10.3389/fpls.2016.00047 A review of auxin response factors (ARFs) in plants Li SY , Zhang X , Wang WS , et al. , 2018 . Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice . Mol Plant , 11 ( 7 ): 995 - 998 . https://doi.org/10.1016/j.molp.2018.03.009 https://doi.org/10.1016/j.molp.2018.03.009 Expanding the scope of CRISPR/Cpf1-mediated genome editing in rice Li TD , Yang XP , Yu Y , et al. , 2018 . Domestication of wild tomato is accelerated by genome editing . Nat Biotechnol , 36 ( 12 ): 1160 - 1163 . https://doi.org/10.1038/nbt.4273 https://doi.org/10.1038/nbt.4273 Domestication of wild tomato is accelerated by genome editing Li XD , Wang YN , Chen S , et al. , 2018 . Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing . Front Plant Sci , 9 : 559 . https://doi.org/10.3389/fpls.2018.00559 https://doi.org/10.3389/fpls.2018.00559 Lycopene is enriched in tomato fruit by CRISPR/Cas9-mediated multiplex genome editing Li XS , Wang Y , Liu YJ , et al. , 2018 . Base editing with a Cpf1-cytidine deaminase fusion . Nat Biotechnol , 36 ( 4 ): 324 - 327 . https://doi.org/10.1038/nbt.4102 https://doi.org/10.1038/nbt.4102 Base editing with a Cpf1-cytidine deaminase fusion Li ZG , 2009 . Comparative study on WHO Western Pacific Region and World Federation of Chinese Medicine Societies international standard terminologies on traditional medicine: an analysis of the Causes of Diseases (Part 1). J Chin Integr Med , 7 ( 3 ): 284 - 287 . https://doi.org/10.3736/jcim20090317 https://doi.org/10.3736/jcim20090317 Comparative study on WHO Western Pacific Region and World Federation of Chinese Medicine Societies international standard terminologies on traditional medicine: an analysis of the Causes of Diseases (Part 1). J Chin Integr Med Li ZJ , Wei JC , Di D , et al. , 2020 . Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay . Acta Biochim Biophys Sin (Shanghai) , 52 ( 12 ): 1413 - 1419 . https://doi.org/10.1093/abbs/gmaa135 https://doi.org/10.1093/abbs/gmaa135 Rapid and accurate detection of African swine fever virus by DNA endonuclease-targeted CRISPR trans reporter assay Liang Z , Zhang K , Chen K L , et al. , 2014 . Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system . J Genet Genomics , 41 ( 2 ): 63 - 68 . https://doi.org/10.1016/j.jgg.2013.12.001 https://doi.org/10.1016/j.jgg.2013.12.001 Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system Liao TT , Lin CC , Jiang JK , et al. , 2020 . Harnessing stemness and PD-L1 expression by AT-rich interaction domain-containing protein 3B in colorectal cancer . Theranostics , 10 ( 14 ): 6095 - 6112 . https://doi.org/10.7150/thno.44147 https://doi.org/10.7150/thno.44147 Harnessing stemness and PD-L1 expression by AT-rich interaction domain-containing protein 3B in colorectal cancer Lin QP , Zong Y , Xue CX , et al. , 2020 . Prime genome editing in rice and wheat . Nat Biotechnol , 38 ( 5 ): 582 - 585 . https://doi.org/10.1038/s41587-020-0455-x https://doi.org/10.1038/s41587-020-0455-x Prime genome editing in rice and wheat Lin S , Staahl BT , Alla RK , et al. , 2014 . Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery . eLife , 3 : e04766 . https://doi.org/10.7554/eLife.04766 https://doi.org/10.7554/eLife.04766 Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery Lin WR , Ng IS , 2020 . Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation . Enzyme Microb Technol , 133 : 109458 . https://doi.org/10.1016/j.enzmictec.2019.109458 https://doi.org/10.1016/j.enzmictec.2019.109458 Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation Liu HW , Zhang BH , 2020 . Virus-based CRISPR/Cas9 genome editing in plants . Trends Genet , 36 ( 11 ): 810 - 813 . https://doi.org/10.1016/j.tig.2020.08.002 https://doi.org/10.1016/j.tig.2020.08.002 Virus-based CRISPR/Cas9 genome editing in plants Liu JW , Sun MY , Cho KB , et al. , 2021 . A CRISPR-Cas9 repressor for epigenetic silencing of KRAS . Pharmacol Res , 164 : 105304 . https://doi.org/10.1016/j.phrs.2020.105304 https://doi.org/10.1016/j.phrs.2020.105304 A CRISPR-Cas9 repressor for epigenetic silencing of KRAS Liu XS , Wu H , Ji X , et al. , 2016 . Editing DNA methylation in the mammalian genome . Cell , 167 ( 1 ): 233 - 247.e17 . https://doi.org/10.1016/j.cell.2016.08.056 https://doi.org/10.1016/j.cell.2016.08.056 Editing DNA methylation in the mammalian genome Liu Y , Merrick P , Zhang ZZ , et al. , 2018 . Targeted mutagenesis in tetraploid switchgrass ( Panicum virgatum L .) using CRISPR/Cas9. Plant Biotechnol J , 16 ( 2 ): 381 - 393 . https://doi.org/10.1111/pbi.12778 https://doi.org/10.1111/pbi.12778 Targeted mutagenesis in tetraploid switchgrass (Panicum virgatum L Liu Y , Wang WL , Yang B , et al. , 2020 . Functional analysis of the teosinte branched 1 gene in the tetraploid switchgrass ( Panicum virgatum L .) by CRISPR/Cas9-directed mutagenesis. Front Plant Sci , 11 : 572193 . https://doi.org/10.3389/fpls.2020.572193 https://doi.org/10.3389/fpls.2020.572193 Functional analysis of the teosinte branched 1 gene in the tetraploid switchgrass (Panicum virgatum L Liu YZ , Qi XW , Zeng ZZ , et al. , 2017 . CRISPR/Cas9-mediated p53 and Pten dual mutation accelerates hepatocarcinogenesis in adult hepatitis B virus transgenic mice . Sci Rep , 7 : 2796 . https://doi.org/10.1038/s41598-017-03070-8 https://doi.org/10.1038/s41598-017-03070-8 CRISPR/Cas9-mediated p53 and Pten dual mutation accelerates hepatocarcinogenesis in adult hepatitis B virus transgenic mice Liu ZQ , Chen M , Chen SY , et al. , 2018 . Highly efficient RNA-guided base editing in rabbit . Nat Commun , 9 : 2717 . https://doi.org/10.1038/s41467-018-05232-2 https://doi.org/10.1038/s41467-018-05232-2 Highly efficient RNA-guided base editing in rabbit Lotfi M , Rezaei N , 2020 . CRISPR/Cas13: a potential therapeutic option of COVID-19 . Biomed Pharmacother , 131 : 110738 . https://doi.org/10.1016/j.biopha.2020.110738 https://doi.org/10.1016/j.biopha.2020.110738 CRISPR/Cas13: a potential therapeutic option of COVID-19 Lu SX , Hu MY , Wang ZH , et al. , 2020 . Generation and application of the zebrafish heg1 mutant as a cardiovascular disease model . Biomolecules , 10 ( 11 ): 1542 . https://doi.org/10.3390/biom10111542 https://doi.org/10.3390/biom10111542 Generation and application of the zebrafish heg1 mutant as a cardiovascular disease model Lu Y , Xue JX , Deng T , et al. , 2020 . Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer . Nat Med , 26 ( 5 ): 732 - 740 . https://doi.org/10.1038/s41591-020-0840-5 https://doi.org/10.1038/s41591-020-0840-5 Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer Lv YS , Shao GN , Jiao GJ , et al. , 2021 . Targeted mutagenesis of POLYAMINE OXIDASE 5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield . Mol Plant , 14 ( 2 ): 344 - 351 . https://doi.org/10.1016/j.molp.2020.11.007 https://doi.org/10.1016/j.molp.2020.11.007 Targeted mutagenesis of POLYAMINE OXIDASE5 that negatively regulates mesocotyl elongation enables the generation of direct-seeding rice with improved grain yield Lyzenga WJ , Harrington M , Bekkaoui D , et al. , 2019 . CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa . BMC Plant Biol , 19 : 292 . https://doi.org/10.1186/s12870-019-1873-0 https://doi.org/10.1186/s12870-019-1873-0 CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa Ma HH , Naseri A , Reyes-Gutierrez P , et al. , 2015 . Multicolor CRISPR labeling of chromosomal loci in human cells . Proc Natl Acad Sci USA , 112 ( 10 ): 3002 - 3007 . https://doi.org/10.1073/pnas.1420024112 https://doi.org/10.1073/pnas.1420024112 Multicolor CRISPR labeling of chromosomal loci in human cells Ma XN , Zhang XY , Liu HM , et al. , 2020 . Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9 . Nat Plants , 6 ( 7 ): 773 - 779 . https://doi.org/10.1038/s41477-020-0704-5 https://doi.org/10.1038/s41477-020-0704-5 Highly efficient DNA-free plant genome editing using virally delivered CRISPR-Cas9 Maddalo D , Manchado E , Concepcion CP , et al. , 2014 . In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system . Nature , 516 ( 7531 ): 423 - 427 . https://doi.org/10.1038/nature13902 https://doi.org/10.1038/nature13902 In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system Maeder ML , Linder SJ , Cascio VM , et al. , 2013 . CRISPR RNA-guided activation of endogenous human genes . Nat Methods , 10 ( 10 ): 977 - 979 . https://doi.org/10.1038/nmeth.2598 https://doi.org/10.1038/nmeth.2598 CRISPR RNA-guided activation of endogenous human genes Magadán AH , Dupuis MÈ , Villion M , et al. , 2012 . Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system . PLoS ONE , 7 ( 7 ): e40913 . https://doi.org/10.1371/journal.pone.0040913 https://doi.org/10.1371/journal.pone.0040913 Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system Mahoney KM , Freeman GJ , McDermott DF , 2015 . The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma . Clin Ther , 37 ( 4 ): 764 - 782 . https://doi.org/10.1016/j.clinthera.2015.02.018 https://doi.org/10.1016/j.clinthera.2015.02.018 The next immune-checkpoint inhibitors: PD-1/PD-L1 blockade in melanoma Makarova KS , Wolf YI , Iranzo J , et al. , 2020 . Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants . Nat Rev Microbiol , 18 ( 2 ): 67 - 83 . https://doi.org/10.1038/s41579-019-0299-x https://doi.org/10.1038/s41579-019-0299-x Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants Malina A , Mills JR , Cencic R , et al. , 2013 . Repurposing CRISPR/Cas9 for in situ functional assays . Genes Dev , 27 ( 23 ): 2602 - 2614 . https://doi.org/10.1101/gad.227132.113 https://doi.org/10.1101/gad.227132.113 Repurposing CRISPR/Cas9 for in situ functional assays Martínez MSI , Bracuto V , Koseoglou E , et al. , 2020 . CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew . BMC Plant Biol , 20 : 284 . https://doi.org/10.1186/s12870-020-02497-y https://doi.org/10.1186/s12870-020-02497-y CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew Meng ZG , Meng ZH , Zhang R , et al. , 2015 . Expression of the rice arginase gene OsARG in cotton influences the morphology and nitrogen transition of seedlings . PLoS ONE , 10 ( 11 ): e0141530 . https://doi.org/10.1371/journal.pone.0141530 https://doi.org/10.1371/journal.pone.0141530 Expression of the rice arginase gene OsARG in cotton influences the morphology and nitrogen transition of seedlings Miller SM , Wang TN , Randolph PB , et al. , 2020 . Continuous evolution of SpCas9 variants compatible with non-G PAMs . Nat Biotechnol , 38 ( 4 ): 471 - 481 . https://doi.org/10.1038/s41587-020-0412-8 https://doi.org/10.1038/s41587-020-0412-8 Continuous evolution of SpCas9 variants compatible with non-G PAMs Mojica FJM , Montoliu L , 2016 . On the origin of CRISPR-Cas technology: from prokaryotes to mammals . Trends Microbiol , 24 ( 10 ): 811 - 820 . https://doi.org/10.1016/j.tim.2016.06.005 https://doi.org/10.1016/j.tim.2016.06.005 On the origin of CRISPR-Cas technology: from prokaryotes to mammals Mojica FJM , Juez G , Rodriguez-Valera F , 1993 . Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified Pst I sites . Mol Microbiol , 9 ( 3 ): 613 - 621 . https://doi.org/10.1111/j.1365-2958.1993.tb01721.x https://doi.org/10.1111/j.1365-2958.1993.tb01721.x Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites Mojica FJM , Ferrer C , Juez G , et al. , 1995 . Long stretches of short tandem repeats are present in the largest replicons of the archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning . Mol Microbiol , 17 : 85 - 93 . https://doi.org/10.1111/j.1365-2958.1995.mmi_17010085.x https://doi.org/10.1111/j.1365-2958.1995.mmi_17010085.x Long stretches of short tandem repeats are present in the largest replicons of the archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning Mojica FJM , Díez-Villaseñor C , García-Martínez J , et al. , 2005 . Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements . J Mol Evol , 60 ( 2 ): 174 - 182 . https://doi.org/10.1007/s00239-004-0046-3 https://doi.org/10.1007/s00239-004-0046-3 Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements Morita S , Noguchi H , Horii T , et al. , 2016 . Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions . Nat Biotechnol , 34 ( 10 ): 1060 - 1065 . https://doi.org/10.1038/nbt.3658 https://doi.org/10.1038/nbt.3658 Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions Müller M , Lee CM , Gasiunas G , et al. , 2016 . Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome . Mol Ther , 24 ( 3 ): 636 - 644 . https://doi.org/10.1038/mt.2015.218 https://doi.org/10.1038/mt.2015.218 Streptococcus thermophilus CRISPR-Cas9 systems enable specific editing of the human genome Mustafa MI , Makhawi AM , 2021 . SHERLOCK and DETECTR: CRISPR-Cas systems as potential rapid diagnostic tools for emerging infectious diseases . J Clin Microbiol , 59 ( 3 ): e00745-20 . https://doi.org/10.1128/jcm.00745-20 https://doi.org/10.1128/jcm.00745-20 SHERLOCK and DETECTR: CRISPR-Cas systems as potential rapid diagnostic tools for emerging infectious diseases Myhrvold C , Freije CA , Gootenberg JS , et al. , 2018 . Field-deployable viral diagnostics using CRISPR-Cas13 . Science , 360 ( 6387 ): 444 - 448 . https://doi.org/10.1126/science.aas8836 https://doi.org/10.1126/science.aas8836 Field-deployable viral diagnostics using CRISPR-Cas13 Nakata A , Amemura M , Makino K , 1989 . Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome . J Bacteriol , 171 ( 6 ): 3553 - 3556 . https://doi.org/10.1128/jb.171.6.3553-3556.1989 https://doi.org/10.1128/jb.171.6.3553-3556.1989 Unusual nucleotide arrangement with repeated sequences in the Escherichiacoli K-12 chromosome Nakayasu M , Akiyama R , Lee HJ , et al. , 2018 . Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene . Plant Physiol Biochem , 131 : 70 - 77 . https://doi.org/10.1016/j.plaphy.2018.04.026 https://doi.org/10.1016/j.plaphy.2018.04.026 Generation of α-solanine-free hairy roots of potato by CRISPR/Cas9 mediated genome editing of the St16DOX gene Nature Methods , 2012 . Method of the year 2011 . Nat Methods , 9 : 1 . https://doi.org/10.1038/nmeth.1852 https://doi.org/10.1038/nmeth.1852 Method of the year 2011 Navet N , Tian MY , 2020 . Efficient targeted mutagenesis in allotetraploid sweet basil by CRISPR/Cas9 . Plant Direct , 4 ( 6 ): e00233 . https://doi.org/10.1002/pld3.233 https://doi.org/10.1002/pld3.233 Efficient targeted mutagenesis in allotetraploid sweet basil by CRISPR/Cas9 Nekrasov V , Wang CM , Win J , et al. , 2017 . Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion . Sci Rep , 7 : 482 . https://doi.org/10.1038/s41598-017-00578-x https://doi.org/10.1038/s41598-017-00578-x Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion Nishida K , Arazoe T , Yachie N , et al. , 2016 . Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems . Science , 353 ( 6305 ): aaf8729 . https://doi.org/10.1126/science.aaf8729 https://doi.org/10.1126/science.aaf8729 Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems Nishimasu H , Cong L , Yan WX , et al. , 2015 . Crystal structure of staphylococcus aureus Cas9 . Cell , 162 ( 5 ): 1113 - 1126 . https://doi.org/10.1016/j.cell.2015.08.007 https://doi.org/10.1016/j.cell.2015.08.007 Crystal structure of staphylococcus aureus Cas9 Nishimasu H , Shi X , Ishiguro S , et al. , 2018 . Engineered CRISPR-Cas9 nuclease with expanded targeting space . Science , 361 ( 6408 ): 1259 - 1262 . https://doi.org/10.1126/science.aas9129 https://doi.org/10.1126/science.aas9129 Engineered CRISPR-Cas9 nuclease with expanded targeting space Nishitani C , Hirai N , Komori S , et al. , 2016 . Efficient genome editing in apple using a CRISPR/Cas9 system . Sci Rep , 6 : 31481 . https://doi.org/10.1038/srep31481 https://doi.org/10.1038/srep31481 Efficient genome editing in apple using a CRISPR/Cas9 system Nonaka S , Arai C , Takayama M , et al. , 2017 . Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis . Sci Rep , 7 : 7057 . https://doi.org/10.1038/s41598-017-06400-y https://doi.org/10.1038/s41598-017-06400-y Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis Odipio J , Alicai T , Ingelbrecht I , et al. , 2017 . Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava . Front Plant Sci , 8 : 1780 . https://doi.org/10.3389/fpls.2017.01780 https://doi.org/10.3389/fpls.2017.01780 Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava Okuzaki A , Ogawa T , Koizuka C , et al. , 2018 . CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus . Plant Physiol Biochem , 131 : 63 - 69 . https://doi.org/10.1016/j.plaphy.2018.04.025 https://doi.org/10.1016/j.plaphy.2018.04.025 CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus Ortigosa A , Gimenez-Ibanez S , Leonhardt N , et al. , 2019 . Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2 . Plant Biotechnol J , 17 ( 3 ): 665 - 673 . https://doi.org/10.1111/pbi.13006 https://doi.org/10.1111/pbi.13006 Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2 Ozseyhan ME , Kang JL , Mu XP , et al. , 2018 . Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa . Plant Physiol Biochem , 123 : 1 - 7 . https://doi.org/10.1016/j.plaphy.2017.11.021 https://doi.org/10.1016/j.plaphy.2017.11.021 Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa Park JJ , Yoo CG , Flanagan A , et al. , 2017 . Defined tetra-allelic gene disruption of the 4-coumarate: coenzyme A ligase 1 ( Pv4CL1 ) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release . Biotechnol Biofuels , 10 : 284 . https://doi.org/10.1186/s13068-017-0972-0 https://doi.org/10.1186/s13068-017-0972-0 Defined tetra-allelic gene disruption of the 4-coumarate: coenzyme A ligase 1 (Pv4CL1) gene by CRISPR/Cas9 in switchgrass results in lignin reduction and improved sugar release Pathi KM , Rink P , Budhagatapalli N , et al. , 2020 . Engineering smut resistance in maize by site-directed mutagenesis of LIPOXYGENASE 3 . Front Plant Sci , 11 : 543895 . https://doi.org/10.3389/fpls.2020.543895 https://doi.org/10.3389/fpls.2020.543895 Engineering smut resistance in maize by site-directed mutagenesis of LIPOXYGENASE 3 Pausch P , Al-Shayeb B , Bisom-Rapp E , et al. , 2020 . CRISPR-CasΦ from huge phages is a hypercompact genome editor . Science , 369 ( 6501 ): 333 - 337 . https://doi.org/10.1126/science.abb1400 https://doi.org/10.1126/science.abb1400 CRISPR-CasΦ from huge phages is a hypercompact genome editor Peng AH , Chen SC , Lei TG , et al. , 2017 . Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus . Plant Biotechnol J , 15 ( 12 ): 1509 - 1519 . https://doi.org/10.1111/pbi.12733 https://doi.org/10.1111/pbi.12733 Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus Peng RH , Jones DC , Liu F , et al. , 2021 . From sequencing to genome editing for cotton improvement . Trends Biotechnol , 39 ( 3 ): 221 - 224 . https://doi.org/10.1016/j.tibtech.2020.09.001 https://doi.org/10.1016/j.tibtech.2020.09.001 From sequencing to genome editing for cotton improvement Pröbsting M , Schenke D , Hossain R , et al. , 2020 . Loss of function of CRT1A (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape ( Brassica napus ) . Plant Biotechnol J , 18 ( 11 ): 2328 - 2344 . https://doi.org/10.1111/pbi.13394 https://doi.org/10.1111/pbi.13394 Loss of function of CRT1A (calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus) Qi LS , Larson MH , Gilbert LA , et al. , 2013 . Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression . Cell , 152 ( 5 ): 1173 - 1183 . https://doi.org/10.1016/j.cell.2013.02.022 https://doi.org/10.1016/j.cell.2013.02.022 Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression Quan J , Langelier C , Kuchta A , et al. , 2019 . FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences . Nucleic Acids Res , 47 ( 14 ): e83 . https://doi.org/10.1093/nar/gkz418 https://doi.org/10.1093/nar/gkz418 FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences Ran FA , Cong L , Yan WX , et al. , 2015 . In vivo genome editing using Staphylococcus aureus Cas9 . Nature , 520 ( 7546 ): 186 - 191 . https://doi.org/10.1038/nature14299 https://doi.org/10.1038/nature14299 In vivo genome editing using Staphylococcus aureus Cas9 Ren B , Liu L , Li SF , et al. , 2019 . Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice . Mol Plant , 12 ( 7 ): 1015 - 1026 . https://doi.org/10.1016/j.molp.2019.03.010 https://doi.org/10.1016/j.molp.2019.03.010 Cas9-NG greatly expands the targeting scope of the genome-editing toolkit by recognizing NG and other atypical PAMs in rice Ren C , Liu XJ , Zhang Z , et al. , 2016 . CRISPR/Cas9-mediated efficient targeted mutagenesis in chardonnay ( Vitis vinifera L . ) . Sci Rep , 6 : 32289 . https://doi.org/10.1038/srep32289 https://doi.org/10.1038/srep32289 CRISPR/Cas9-mediated efficient targeted mutagenesis in chardonnay (Vitis vinifera L Richter MF , Zhao KT , Eton E , et al. , 2020 . Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity . Nat Biotechnol , 38 : 883 - 891 . https://doi.org/10.1038/s41587-020-0453-z https://doi.org/10.1038/s41587-020-0453-z Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity Roca Paixão JF , Gillet FX , Ribeiro TP , et al. , 2019 . Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a histone acetyltransferase . Sci Rep , 9 : 8080 . https://doi.org/10.1038/s41598-019-44571-y https://doi.org/10.1038/s41598-019-44571-y Improved drought stress tolerance in Arabidopsis by CRISPR/dCas9 fusion with a histone acetyltransferase Ron M , Kajala K , Pauluzzi G , et al. , 2014 . Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model . Plant Physiol , 166 ( 2 ): 455 - 469 . https://doi.org/10.1104/pp.114.239392 https://doi.org/10.1104/pp.114.239392 Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model Sánchez-León S , Gil-Humanes J , Ozuna CV , et al. , 2018 . Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9 . Plant Biotechnol J , 16 ( 4 ): 902 - 910 . https://doi.org/10.1111/pbi.12837 https://doi.org/10.1111/pbi.12837 Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9 Santillán Martínez MI , Bracuto V , Koseoglou E , et al. , 2020 . CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew . BMC Plant Biol , 20 : 284 . https://doi.org/10.1186/s12870-020-02497-y https://doi.org/10.1186/s12870-020-02497-y CRISPR/Cas9-targeted mutagenesis of the tomato susceptibility gene PMR4 for resistance against powdery mildew Sashidhar N , Harloff HJ , Potgieter L , et al. , 2020 . Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds . Plant Biotechnol J , 18 ( 11 ): 2241 - 2250 . https://doi.org/10.1111/pbi.13380 https://doi.org/10.1111/pbi.13380 Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds Sauer NJ , Narváez-Vásquez J , Mozoruk J , et al. , 2016 . Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants . Plant Physiol , 170 ( 4 ): 1917 - 1928 . https://doi.org/10.1104/pp.15.01696 https://doi.org/10.1104/pp.15.01696 Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants Shan QW , Wang YP , Li J , et al. , 2013 . Targeted genome modification of crop plants using a CRISPR-Cas system . Nat Biotechnol , 31 ( 8 ): 686 - 688 . https://doi.org/10.1038/nbt.2650 https://doi.org/10.1038/nbt.2650 Targeted genome modification of crop plants using a CRISPR-Cas system Shi Y , Wang G , Cai XP , et al. , 2020 . An overview of COVID-19 . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 21 ( 5 ): 343 - 360 . https://doi.org/10.1631/jzus.B2000083 https://doi.org/10.1631/jzus.B2000083 An overview of COVID-19 Shin JJ , Schröder MS , Caiado F , et al. , 2020 . Controlled cycling and quiescence enables efficient HDR in engraftment-enriched adult hematopoietic stem and progenitor cells . Cell Rep , 32 ( 9 ): 108093 . https://doi.org/10.1016/j.celrep.2020.108093 https://doi.org/10.1016/j.celrep.2020.108093 Controlled cycling and quiescence enables efficient HDR in engraftment-enriched adult hematopoietic stem and progenitor cells Silva G , Poirot L , Galetto R , et al. , 2011 . Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy . Curr Gene Ther , 11 ( 1 ): 11 - 27 . https://doi.org/10.2174/156652311794520111 https://doi.org/10.2174/156652311794520111 Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy Soares F , Chen B , Lee JB , et al. , 2020 . CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy . Blood , online . https://doi.org/10.1182/blood.2019004108 https://doi.org/10.1182/blood.2019004108 CRISPR screen identifies genes that sensitize AML cells to double negative T cell therapy Stadtmauer EA , Fraietta JA , Davis MM , et al. , 2020 . CRISPR-engineered T cells in patients with refractory cancer . Science , 367 ( 6481 ): eaba7365 . https://doi.org/10.1126/science.aba7365 https://doi.org/10.1126/science.aba7365 CRISPR-engineered T cells in patients with refractory cancer Steinert J , Schiml S , Fauser F , et al. , 2015 . Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus . Plant J , 84 ( 6 ): 1295 - 1305 . https://doi.org/10.1111/tpj.13078 https://doi.org/10.1111/tpj.13078 Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus Strahl BD , Allis CD , 2000 . The language of covalent histone modifications . Nature , 403 ( 6765 ): 41 - 45 . https://doi.org/10.1038/47412 https://doi.org/10.1038/47412 The language of covalent histone modifications Su S , Zou ZY , Chen FJ , et al. , 2017 . CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer . Oncoimmunology , 6 ( 1 ): e1249558 . https://doi.org/10.1080/2162402x.2016.1249558 https://doi.org/10.1080/2162402x.2016.1249558 CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer Sun B , Jiang M , Zheng H , et al. , 2020 . Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO . Hortic Res , 7 : 161 . https://doi.org/10.1038/s41438-020-00379-w https://doi.org/10.1038/s41438-020-00379-w Color-related chlorophyll and carotenoid concentrations of Chinese kale can be altered through CRISPR/Cas9 targeted editing of the carotenoid isomerase gene BoaCRTISO Sun N , Petiwala S , Wang R , et al. , 2019 . Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening . BMC Genomics , 20 : 225 . https://doi.org/10.1186/s12864-019-5601-9 https://doi.org/10.1186/s12864-019-5601-9 Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening Sun YW , Jiao GA , Liu ZP , et al. , 2017 . Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes . Front Plant Sci , 8 : 298 . https://doi.org/10.3389/fpls.2017.00298 https://doi.org/10.3389/fpls.2017.00298 Generation of high-amylose rice through CRISPR/Cas9-mediated targeted mutagenesis of starch branching enzymes Sweeney MT , Thomson MJ , Cho YG , et al. , 2007 . Global dissemination of a single mutation conferring white pericarp in rice . PLoS Genet , 3 ( 8 ): e133 . https://doi.org/10.1371/journal.pgen.0030133 https://doi.org/10.1371/journal.pgen.0030133 Global dissemination of a single mutation conferring white pericarp in rice Syombua ED , Zhang ZZ , Tripathi JN , et al. , 2020 . A CRISPR/Cas9-based genome-editing system for yam (Dioscorea spp . . Plant Biotechnol J , online . https://doi.org/10.1111/pbi.13515 https://doi.org/10.1111/pbi.13515 Plant Biotechnol J, online Takahashi N , Cho P , Selfors LM , et al. , 2020 . 3D culture models with CRISPR screens reveal hyperactive NRF2 as a prerequisite for spheroid formation via regulation of proliferation and ferroptosis . Mol Cell , 80 ( 5 ): 828 - 844.e6 . https://doi.org/10.1016/j.molcel.2020.10.010 https://doi.org/10.1016/j.molcel.2020.10.010 3D culture models with CRISPR screens reveal hyperactive NRF2 as a prerequisite for spheroid formation via regulation of proliferation and ferroptosis Tan JJ , Zhang F , Karcher D , et al. , 2020 . Expanding the genome-targeting scope and the site selectivity of high-precision base editors . Nat Commun , 11 : 629 . https://doi.org/10.1038/s41467-020-14465-z https://doi.org/10.1038/s41467-020-14465-z Expanding the genome-targeting scope and the site selectivity of high-precision base editors Tan YY , Du H , Wu X , et al. , 2020 . Gene editing: an instrument for practical application of gene biology to plant breeding . J Zhejiang Univ-Sci B (Biomed & Biotechnol) , 21 ( 6 ): 460 - 473 . https://doi.org/10.1631/jzus.B1900633 https://doi.org/10.1631/jzus.B1900633 Gene editing: an instrument for practical application of gene biology to plant breeding Tanenbaum ME , Gilbert LA , Qi LS , et al. , 2014 . A protein-tagging system for signal amplification in gene expression and fluorescence imaging . Cell , 159 ( 3 ): 635 - 646 . https://doi.org/10.1016/j.cell.2014.09.039 https://doi.org/10.1016/j.cell.2014.09.039 A protein-tagging system for signal amplification in gene expression and fluorescence imaging Tang X , Lowder LG , Zhang T , et al. , 2017 . A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants . Nat Plants , 3 ( 3 ): 17018 . https://doi.org/10.1038/Nplants.2017.18 https://doi.org/10.1038/Nplants.2017.18 A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants Tang X , Sretenovic S , Ren QR , et al. , 2020 . Plant prime editors enable precise gene editing in rice cells . Mol Plant , 13 ( 5 ): 667 - 670 . https://doi.org/10.1016/j.molp.2020.03.010 https://doi.org/10.1016/j.molp.2020.03.010 Plant prime editors enable precise gene editing in rice cells Tian XL , Gu TX , Patel S , et al. , 2019 . CRISPR/Cas9—an evolving biological tool kit for cancer biology and oncology . NPJ Precis Oncol , 3 : 8 . https://doi.org/10.1038/s41698-019-0080-7 https://doi.org/10.1038/s41698-019-0080-7 CRISPR/Cas9—an evolving biological tool kit for cancer biology and oncology Tian YS , Chen K , Li X , et al. , 2020 . Design of high-oleic tobacco ( Nicotiana tabacum L .) seed oil by CRISPR-Cas9-mediated knockout of NtFAD2 - 2 . BMC Plant Biol , 20 : 233 . https://doi.org/10.1186/s12870-020-02441-0 https://doi.org/10.1186/s12870-020-02441-0 Design of high-oleic tobacco (Nicotiana tabacum L Travis J , 2015 . Makes the cut: CRISPR genome-editing technology shows its power . Science , 350 ( 6267 ): 1456 - 1457 . https://doi.org/10.1126/science.350.6267.1456 https://doi.org/10.1126/science.350.6267.1456 Makes the cut: CRISPR genome-editing technology shows its power van den Broeck HC , van Herpen TWJM , Schuit C , et al. , 2009 . Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese spring deletion lines . BMC Plant Biol , 9 : 41 . https://doi.org/10.1186/1471-2229-9-41 https://doi.org/10.1186/1471-2229-9-41 Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese spring deletion lines Vojta A , Dobrinić P , Tadić V , et al. , 2016 . Repurposing the CRISPR-Cas9 system for targeted DNA methylation . Nucleic Acids Res , 44 ( 12 ): 5615 - 5628 . https://doi.org/10.1093/nar/gkw159 https://doi.org/10.1093/nar/gkw159 Repurposing the CRISPR-Cas9 system for targeted DNA methylation Walton RT , Christie KA , Whittaker MN , et al. , 2020 . Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants . Science , 368 ( 6488 ): 290 - 296 . https://doi.org/10.1126/science.aba8853 https://doi.org/10.1126/science.aba8853 Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants Wan DY , Guo Y , Cheng Y , et al. , 2020 . CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine ( Vitis vinifera ) . Hortic Res , 7 : 116 . https://doi.org/10.1038/s41438-020-0339-8 https://doi.org/10.1038/s41438-020-0339-8 CRISPR/Cas9-mediated mutagenesis of VvMLO3 results in enhanced resistance to powdery mildew in grapevine (Vitis vinifera) Wang FJ , Wang CL , Liu PQ , et al. , 2016 . Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922 . PLoS ONE , 11 ( 4 ): e0154027 . https://doi.org/10.1371/journal.pone.0154027 https://doi.org/10.1371/journal.pone.0154027 Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922 Wang HX , Wu YL , Zhang YD , et al. , 2019 . CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato ( Ipomoea batatas ) for the improvement of starch quality . Int J Mol Sci , 20 ( 19 ): 4702 . CRISPR/Cas9-based mutagenesis of starch biosynthetic genes in sweet potato (Ipomoea batatas) for the improvement of starch quality Wang JJ , Meng XB , Hu XX , et al. , 2019 . xCas9 expands the scope of genome editing with reduced efficiency in rice . Plant Biotechnol J , 17 ( 4 ): 709 - 711 . https://doi.org/10.1111/pbi.13053 https://doi.org/10.1111/pbi.13053 xCas9 expands the scope of genome editing with reduced efficiency in rice Wang LJ , Chen SC , Peng AH , et al. , 2019 . CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonas citri subsp . citri in Wanjincheng orange (Citrus sinensis (L.) Osbeck). Plant Biotechnol Rep , 13 ( 5 ): 501 - 510 . https://doi.org/10.1007/s11816-019-00556-x https://doi.org/10.1007/s11816-019-00556-x CRISPR/Cas9-mediated editing of CsWRKY22 reduces susceptibility to Xanthomonascitri subsp Wang M , Zhang R , Li JM , 2020 . CRISPR/Cas systems redefine nucleic acid detection: principles and methods . Biosens Bioelectron , 165 : 112430 . https://doi.org/10.1016/j.bios.2020.112430 https://doi.org/10.1016/j.bios.2020.112430 CRISPR/Cas systems redefine nucleic acid detection: principles and methods Wang SH , Zhang SB , Wang WX , et al. , 2015 . Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system . Plant Cell Rep , 34 ( 9 ): 1473 - 1476 . https://doi.org/10.1007/s00299-015-1816-7 https://doi.org/10.1007/s00299-015-1816-7 Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system Wang WT , Wang ZL , Hou WS , et al. , 2020 . GmNMHC5 , a neoteric positive transcription factor of flowering and maturity in soybean . Plants (Basel) , 9 ( 6 ): 792 . https://doi.org/10.3390/plants9060792 https://doi.org/10.3390/plants9060792 GmNMHC5, a neoteric positive transcription factor of flowering and maturity in soybean Wang XJ , Shang XY , Huang XX , 2020a . Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods . Emerg Microbes Infect , 9 ( 1 ): 1682 - 1691 . https://doi.org/10.1080/22221751.2020.1793689 https://doi.org/10.1080/22221751.2020.1793689 Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods Wang XJ , Zhong MT , Liu Y , et al. , 2020b . Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER . Sci Bull (Beijing) , 65 ( 17 ): 1436 - 1439 . https://doi.org/10.1016/j.scib.2020.04.041 https://doi.org/10.1016/j.scib.2020.04.041 Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER Wang YL , Meng ZG , Liang CZ , et al. , 2017 . Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton . Sci China Life Sci , 60 ( 5 ): 524 - 527 . https://doi.org/10.1007/s11427-017-9031-y https://doi.org/10.1007/s11427-017-9031-y Increased lateral root formation by CRISPR/Cas9-mediated editing of arginase genes in cotton Wang YP , Cheng X , Shan QW , et al. , 2014 . Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew . Nat Biotechnol , 32 ( 9 ): 947 - 951 . https://doi.org/10.1038/nbt.2969 https://doi.org/10.1038/nbt.2969 Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew Wu MJ , Liu HQ , Lin Y , et al. , 2020 . In-frame and frame-shift editing of the Ehd1 gene to develop Japonica rice with prolonged basic vegetative growth periods . Front Plant Sci , 11 : 307 . https://doi.org/10.3389/fpls.2020.00307 https://doi.org/10.3389/fpls.2020.00307 In-frame and frame-shift editing of the Ehd1 gene to develop Japonica rice with prolonged basic vegetative growth periods Wu XT , Mao SQ , Ying YC , et al. , 2019 . Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms . Genom Proteom Bioinf , 17 ( 2 ): 119 - 128 . https://doi.org/10.1016/j.gpb.2018.10.001 https://doi.org/10.1016/j.gpb.2018.10.001 Progress and challenges for live-cell imaging of genomic loci using CRISPR-based platforms Wu Y , Xu W , Wang FP , et al. , 2019 . Increasing cytosine base editing scope and efficiency with engineered Cas9-pmCDA1 fusions and the modified sgRNA in rice . Front Genet , 10 : 379 . https://doi.org/10.3389/Fgene.2019.00379 https://doi.org/10.3389/Fgene.2019.00379 Increasing cytosine base editing scope and efficiency with engineered Cas9-pmCDA1 fusions and the modified sgRNA in rice Xie HH , Ge XL , Yang FY , et al. , 2020 . High-fidelity SaCas9 identified by directional screening in human cells . PLoS Biol , 18 ( 7 ): e3000747 . https://doi.org/10.1371/journal.pbio.3000747 https://doi.org/10.1371/journal.pbio.3000747 High-fidelity SaCas9 identified by directional screening in human cells Xie T , Chen X , Guo T , et al. , 2020 . Targeted knockout of BnTT2 homologues for yellow-seeded Brassica napus with reduced flavonoids and improved fatty acid composition . J Agric Food Chem , 68 ( 20 ): 5676 - 5690 . https://doi.org/10.1021/acs.jafc.0c01126 https://doi.org/10.1021/acs.jafc.0c01126 Targeted knockout of BnTT2 homologues for yellow-seeded Brassica napus with reduced flavonoids and improved fatty acid composition Xu JY , Lee YK , Wang Y , et al. , 2014 . Therapeutic application of endothelial progenitor cells for treatment of cardiovascular diseases . Curr Stem Cell Res Ther , 9 ( 5 ): 401 - 414 . https://doi.org/10.2174/1574888x09666140619121318 https://doi.org/10.2174/1574888x09666140619121318 Therapeutic application of endothelial progenitor cells for treatment of cardiovascular diseases Xu L , Wang J , Liu YL , et al. , 2019 . CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia . N Engl J Med , 381 ( 13 ): 1240 - 1247 . https://doi.org/10.1056/NEJMoa1817426 https://doi.org/10.1056/NEJMoa1817426 CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia Xu RF , Yang YC , Qin RY , et al. , 2016 . Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice . J Genet Genomics , 43 ( 8 ): 529 - 532 . https://doi.org/10.1016/j.jgg.2016.07.003 https://doi.org/10.1016/j.jgg.2016.07.003 Rapid improvement of grain weight via highly efficient CRISPR/Cas9-mediated multiplex genome editing in rice Xu RF , Qin RY , Li H , et al. , 2017 . Generation of targeted mutant rice using a CRISPR-Cpf1 system . Plant Biotechnol J , 15 ( 6 ): 713 - 717 . https://doi.org/10.1111/pbi.12669 https://doi.org/10.1111/pbi.12669 Generation of targeted mutant rice using a CRISPR-Cpf1 system Xu W , Zhang CW , Yang YX , et al. , 2020 . Versatile nucleotides substitution in plant using an improved prime editing system . Mol Plant , 13 ( 5 ): 675 - 678 . https://doi.org/10.1016/j.molp.2020.03.012 https://doi.org/10.1016/j.molp.2020.03.012 Versatile nucleotides substitution in plant using an improved prime editing system Xue W , Chen SD , Yin H , et al. , 2014 . CRISPR-mediated direct mutation of cancer genes in the mouse liver . Nature , 514 ( 7522 ): 380 - 384 . https://doi.org/10.1038/nature13589 https://doi.org/10.1038/nature13589 CRISPR-mediated direct mutation of cancer genes in the mouse liver Yahata T , Mizoguchi M , Kimura A , et al. , 2019 . Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression . Cancer Sci , 110 ( 4 ): 1279 - 1292 . https://doi.org/10.1111/cas.13958 https://doi.org/10.1111/cas.13958 Programmed cell death ligand 1 disruption by clustered regularly interspaced short palindromic repeats/Cas9-genome editing promotes antitumor immunity and suppresses ovarian cancer progression Yang H , Wang HY , Jaenisch R , 2014 . Generating genetically modified mice using CRISPR/Cas-mediated genome engineering . Nat Protoc , 9 ( 8 ): 1956 - 1968 . https://doi.org/10.1038/nprot.2014.134 https://doi.org/10.1038/nprot.2014.134 Generating genetically modified mice using CRISPR/Cas-mediated genome engineering Yang XY , Chen L , He JX , et al. , 2017 . Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis . Plant Cell Rep , 36 ( 10 ): 1533 - 1545 . https://doi.org/10.1007/s00299-017-2172-6 https://doi.org/10.1007/s00299-017-2172-6 Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis Yim YY , Teague CD , Nestler EJ , 2020 . In vivo locus-specific editing of the neuroepigenome . Nat Rev Neurosci , 21 ( 9 ): 471 - 484 . https://doi.org/10.1038/s41583-020-0334-y https://doi.org/10.1038/s41583-020-0334-y In vivo locus-specific editing of the neuroepigenome Yu QH , Wang BK , Li N , et al. , 2017 . CRISPR/Cas9-induced targeted mutagenesis and gene replacement to generate long-shelf life tomato lines . Sci Rep , 7 : 11874 . https://doi.org/10.1038/s41598-017-12262-1 https://doi.org/10.1038/s41598-017-12262-1 CRISPR/Cas9-induced targeted mutagenesis and gene replacement to generate long-shelf life tomato lines Yuan M , Zhu J , Gong LM , et al. , 2019 . Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing . BMC Biotechnol , 19 : 24 . https://doi.org/10.1186/s12896-019-0516-8 https://doi.org/10.1186/s12896-019-0516-8 Mutagenesis of FAD2 genes in peanut with CRISPR/Cas9 based gene editing Zeballos CMA , Gaj T , 2020 . Next-generation CRISPR technologies and their applications in gene and cell therapy . Trends Biotechnol , online . https://doi.org/10.1016/j.tibtech.2020.10.010 https://doi.org/10.1016/j.tibtech.2020.10.010 Next-generation CRISPR technologies and their applications in gene and cell therapy Zetsche B , Gootenberg JS , Abudayyeh OO , et al. , 2015 . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . Cell , 163 ( 3 ): 759 - 771 . https://doi.org/10.1016/j.cell.2015.09.038 https://doi.org/10.1016/j.cell.2015.09.038 Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system Zhai YG , Yu KD , Cai SL , et al. , 2020 . Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L . Plant Biotechnol J , 18 ( 5 ): 1153 - 1168 . https://doi.org/10.1111/pbi.13281 https://doi.org/10.1111/pbi.13281 Targeted mutagenesis of BnTT8 homologs controls yellow seed coat development for effective oil production in Brassica napus L Zhang BH , 2021 . CRISPR/Cas gene therapy . J Cell Physiol , 236 ( 4 ): 2459 - 2481 . https://doi.org/10.1002/jcp.30064 https://doi.org/10.1002/jcp.30064 CRISPR/Cas gene therapy Zhang BH , Pan XP , Cobb GP , et al. , 2007a . MicroRNAs as oncogenes and tumor suppressors . Dev Biol , 302 ( 1 ): 1 - 12 . https://doi.org/10.1016/j.ydbio.2006.08.028 https://doi.org/10.1016/j.ydbio.2006.08.028 MicroRNAs as oncogenes and tumor suppressors Zhang BH , Wang QL , Pan XP , 2007b . MicroRNAs and their regulatory roles in animals and plants . J Cell Physiol , 210 ( 2 ): 279 - 289 . https://doi.org/10.1002/jcp.20869 https://doi.org/10.1002/jcp.20869 MicroRNAs and their regulatory roles in animals and plants Zhang BH , Wang QL , Liu F , et al. , 2009 . Highly efficient plant regeneration through somatic embryogenesis in 20 elite commercial cotton ( Gossypium hirsutum L.) cultivars . Plant Omics J , 2 ( 6 ): 259 - 268 . Highly efficient plant regeneration through somatic embryogenesis in 20 elite commercial cotton (Gossypium hirsutum L.) cultivars Zhang DQ , Zhang BH , 2020a . Pectin drives cell wall morphogenesis without turgor pressure . Trends Plant Sci , 25 ( 8 ): 719 - 722 . https://doi.org/10.1016/j.tplants.2020.05.007 https://doi.org/10.1016/j.tplants.2020.05.007 Pectin drives cell wall morphogenesis without turgor pressure Zhang DQ , Zhang BH , 2020b . SpRY: engineered CRISPR/Cas9 harnesses new genome-editing power . Trends Genet , 36 ( 8 ): 546 - 548 . https://doi.org/10.1016/j.tig.2020.05.001 https://doi.org/10.1016/j.tig.2020.05.001 SpRY: engineered CRISPR/Cas9 harnesses new genome-editing power Zhang DQ , Zhang ZY , Unver T , et al. , 2021 . CRISPR/Cas: a powerful tool for gene function study and crop improvement . J Adv Res , 29 : 207 - 221 . https://doi.org/10.1016/j.jare.2020.10.003 https://doi.org/10.1016/j.jare.2020.10.003 CRISPR/Cas: a powerful tool for gene function study and crop improvement Zhang HM , Lang ZB , Zhu JK , 2018 . Dynamics and function of DNA methylation in plants . Nat Rev Mol Cell Biol , 19 ( 8 ): 489 - 506 . https://doi.org/10.1038/s41580-018-0016-z https://doi.org/10.1038/s41580-018-0016-z Dynamics and function of DNA methylation in plants Zhang JS , Zhang H , Botella JR , et al. , 2018 . Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties . J Integr Plant Biol , 60 ( 5 ): 369 - 375 . https://doi.org/10.1111/jipb.12620 https://doi.org/10.1111/jipb.12620 Generation of new glutinous rice by CRISPR/Cas9-targeted mutagenesis of the Waxy gene in elite rice varieties Zhang K , Nie LL , Cheng QQ , et al. , 2019 . Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed ( Brassica napus L .) using CRISPR-Cas9 system. Biotechnol Biofuels , 12 : 225 . https://doi.org/10.1186/s13068-019-1567-8 https://doi.org/10.1186/s13068-019-1567-8 Effective editing for lysophosphatidic acid acyltransferase 2/5 in allotetraploid rapeseed (Brassica napus L Zhang M , Liu QL , Yang XP , et al. , 2020 . CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f . sp. niveum. Plant Cell Rep , 39 ( 5 ): 589 - 595 . https://doi.org/10.1007/s00299-020-02516-0 https://doi.org/10.1007/s00299-020-02516-0 CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f Zhang Y , Li D , Zhang DB , et al. , 2018 . Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits . Plant J , 94 ( 5 ): 857 - 866 . https://doi.org/10.1111/tpj.13903 https://doi.org/10.1111/tpj.13903 Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits Zhang ZN , Ge XY , Luo XY , et al. , 2018 . Simultaneous editing of two copies of Gh14 - 3 - 3d confers enhanced transgene-clean plant defense against Verticillium dahliae in allotetraploid upland cotton . Front Plant Sci , 9 : 842 . https://doi.org/10.3389/fpls.2018.00842 https://doi.org/10.3389/fpls.2018.00842 3d confers enhanced transgene-clean plant defense against Verticillium dahliae in allotetraploid upland cotton Zhao DD , Li J , Li SW , et al. , 2020 . Glycosylase base editors enable C-to-A and C-to-G base changes . Nat Biotechnol , 39 ( 1 ): 35 - 40 . https://doi.org/10.1038/s41587-020-0592-2 https://doi.org/10.1038/s41587-020-0592-2 Glycosylase base editors enable C-to-A and C-to-G base changes Zheng M , Zhang L , Tang M , et al. , 2020 . Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed ( Brassica napus L . ) . Plant Biotechnol J , 18 ( 3 ): 644 - 654 . https://doi.org/10.1111/pbi.13228 https://doi.org/10.1111/pbi.13228 Knockout of two BnaMAX1 homologs by CRISPR/Cas9-targeted mutagenesis improves plant architecture and increases yield in rapeseed (Brassica napus L Zhong ZH , Zhang YX , You Q , et al. , 2018 . Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites . Mol Plant , 11 ( 7 ): 999 - 1002 . https://doi.org/10.1016/j.molp.2018.03.008 https://doi.org/10.1016/j.molp.2018.03.008 Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites Zhong ZH , Sretenovic S , Ren QR , et al. , 2019 . Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG . Mol Plant , 12 ( 7 ): 1027 - 1036 . https://doi.org/10.1016/j.molp.2019.03.011 https://doi.org/10.1016/j.molp.2019.03.011 Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG Zhou XH , Jacobs TB , Xue LJ , et al. , 2015 . Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4-coumarate:CoA ligase specificity and redundancy . New Phytol , 208 ( 2 ): 298 - 301 . https://doi.org/10.1111/nph.13470 https://doi.org/10.1111/nph.13470 Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4-coumarate:CoA ligase specificity and redundancy Zhou Y , Zhao WW , Lai Y , et al. , 2020 . Edible plant oil: global status, health issues, and perspectives . Front Plant Sci , 11 : 1315 . https://doi.org/10.3389/fpls.2020.01315 https://doi.org/10.3389/fpls.2020.01315 Edible plant oil: global status, health issues, and perspectives Zhu YW , Lin YR , Chen SB , et al. , 2019 . CRISPR/Cas9-mediated functional recovery of the recessive rc allele to develop red rice . Plant Biotechnol J , 17 ( 11 ): 2096 - 2105 . https://doi.org/10.1111/pbi.13125 https://doi.org/10.1111/pbi.13125 CRISPR/Cas9-mediated functional recovery of the recessive rc allele to develop red rice Zong Y , Song QN , Li C , et al. , 2018 . Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A . Nat Biotechnol , 36 ( 10 ): 950 - 953 . https://doi.org/10.1038/nbt.4261 https://doi.org/10.1038/nbt.4261 Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A Zsögön A , Čermák T , Naves ER , et al. , 2018 . De novo domestication of wild tomato using genome editing . Nat Biotechnol , 36 ( 12 ): 1211 - 1216 . https://doi.org/10.1038/nbt.4272 https://doi.org/10.1038/nbt.4272 De novo domestication of wild tomato using genome editing Zuo JR , Li JY , 2014 . Molecular genetic dissection of quantitative trait loci regulating rice grain size . Annu Rev Genet , 48 : 99 - 118 . https://doi.org/10.1146/annurev-genet-120213-092138 https://doi.org/10.1146/annurev-genet-120213-092138 Molecular genetic dissection of quantitative trait loci regulating rice grain size All authors were actively involved in summarizing the literature and writing the manuscript. All authors have read and approved the final manuscript. We greatly appreciate the scientific community for making such rapid progress in this field. We apologize to authors whose wonderful work was not cited in this paper. Baohong ZHANG is supported in part by Cotton Incorporated and the National Science Foundation (award 1658709). This work was supported by the National Natural Science Foundation of China (No. 31700316), the Fundamental Research Funds for the Central Nonprofit Scientific Institution (No. 1610172018009), and the Natural Science Foundation of Hubei Province (No. 2018CFB543), China. 10.1631/jzus.B2100009 1673-1581（2021）04-0253-32 2021-01-04 2021-01-27 2021-03-11 2021-04-15 2022-05-09 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2021 2021 浙江大学出版社 Chao LI, Eleanor BRANT, Hikmet BUDAK, and Baohong ZHANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 4 22