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

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. 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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