CRISPR-CAS系统检测传染病病原体的研究进展

Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens CRISPR-CAS系统检测传染病病原体的研究进展 1 equal first-author 1 2 equal https://orcid.org/0000-0001-9062-0999 3 1 corresp Dongli MA, madl1234@126.com Dongli MA, madl1234@126.com madl1234@126.com https://orcid.org/0000-0001-5299-6207 Institute of Pediatrics, Shenzhen Children’s Hospital, Shenzhen 518026, China Institute of Pediatrics, Shenzhen Children’s Hospital, Shenzhen 518026, China 深圳市儿童医院儿科研究所，中国深圳，518026 深圳市儿童医院儿科研究所，中国深圳，518026 CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 中国科学院微生物研究所病原微生物学重点实验室，中国北京，100101 中国科学院微生物研究所病原微生物学重点实验室，中国北京，100101 Frontiers Science Center for Flexible Electronics (FSCFE), Xi’an Institute of Flexible Electronics (IFE) & Xi’an Institute of Biomedical Materials and Engineering (IBME), Northwestern Polytechnical University (NPU), Xi’an 710072, China Frontiers Science Center for Flexible Electronics (FSCFE), Xi’an Institute of Flexible Electronics (IFE) & Xi’an Institute of Biomedical Materials and Engineering (IBME), Northwestern Polytechnical University (NPU), Xi’an 710072, China 西北工业大学柔性电子研究院，中国西安，710072 西北工业大学柔性电子研究院，中国西安，710072 Infectious diseases cause great economic loss and individual and even social anguish. Existing detection methods lack sensitivity and specificity, have a poor turnaround time, and are dependent on expensive equipment. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)‍-CRISPR-associated protein (Cas) system has been widely used in the detection of pathogens that cause infectious diseases owing to its high specificity, sensitivity, and speed, and good accessibility. In this review, we discuss the discovery and development of the CRISPR-Cas system, summarize related analysis and interpretation methods, and discuss the existing applications of CRISPR-based detection of infectious pathogens using Cas proteins. We conclude the challenges and prospects of the CRISPR-Cas system in the detection of pathogens. 感染性疾病对个人乃至社会均可造成极大的经济损失和精神痛苦。现有检测方法在灵敏度与特异性、检测时间、昂贵仪器设备依赖方面或多或少存在不足。成簇的规律间隔的短回文重复序列（CRISPR）-相关蛋白（Cas）系统，因其具备特异、灵敏、快速、便捷等优势，近年来被尝试应用于感染性疾病病原体的检测。本文首先回顾了CRISPR-Cas系统的发现发展进程，并列举可与之结合获得检测结果的检测结果阅读方式，之后从检测过程中所应用的不同Cas蛋白的角度简要描述了目前已有的基于CRISRP检测感染性疾病病原体的应用，最后还展望了CRISPR/Cas系统未来在检测方面的应用前景及挑战。 CRISPR-Cas Diagnosis Disease detection Infectious disease Pathogen CRISPR-Cas 诊断 疾病检测 传染病 病原体 1 Introduction 1 Infectious diseases caused by viruses, bacteria, parasites, fungi, and other pathogenic microorganisms are estimated to cause more than 13 million deaths each year ( Yu et al., 2012 Yu et al., 2012 ; Hwang et al., 2018 Hwang et al., 2018 ). Among these microorganisms, bacteria and viruses have rapid mutation rates and strong transmission abilities, posing severe threats to humans. These diseases often cause great economic losses and social disruption ( Lozano et al., 2012 Lozano et al., 2012 ). Detecting these pathogenic microorganisms in a timely manner is important to prevent the spread of infectious diseases ( Kostyusheva et al., 2022 Kostyusheva et al., 2022 ). Microbial culture methods were among the first diagnostic techniques for infectious diseases and have become the standard for the detection and identification of pathogens. However, microbial culture methods require a long incubation period, and some pathogens may not be easy to culture ( Scheler et al., 2014 Scheler et al., 2014 ). Molecular-level detection methods (including diagnosis through analysis of pathogen-derived antigens, the patient immune response, or the presence of nucleic acid substances) are widely used in pathogen detection ( Taylor et al., 2017 Taylor et al., 2017 ). Antigen-based detection is effective and rapid, but requires preproduction of antibodies and lacks accuracy and sensitivity. Although serological analyses and other adaptive immune response tests are well-developed, they cannot be used for the early diagnosis of infection. Early diagnosis of infection is critical to control the spread of diseases because it can identify the source of infection and accelerate the adoption of anti-infection measures. Existing nucleic acid detection methods adopted for the diagnosis of infectious diseases include polymerase chain reaction (PCR), isothermal amplification, and whole genome sequencing. PCR, the most common detection method, involves nucleic acid amplification for the quantitative analysis of pathogen nucleic acids (including DNA and RNA). PCR is highly sensitive, but its wide application is limited by the need for special equipment and personnel. Isothermal amplification technology is time-saving and portable, but nonspecific amplification has poor accuracy and produces false-positive results. Whole genome sequencing is highly accurate, but is expensive, time-consuming, and complex, rendering it unsuitable for rapid diagnosis of infectious diseases ( Scheler et al., 2014 Scheler et al., 2014 ; Zhao et al., 2015 Zhao et al., 2015 ; Matthijs et al., 2016 Matthijs et al., 2016 ; Taylor et al., 2017 Taylor et al., 2017 ). The World Health Organization (WHO) has proposed that ideal methods for detecting pathogens should be quick, specific, sensitive, and cost-effective, and not require instruments and equipment ( Wang et al., 2020b Wang et al., 2020b ). The development of such methods is imperative in the wake of an increase in the number of pathogens. In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has been used not only for genome and ribonucleic acid editing, but also for nucleic acid detection ( Gootenberg et al., 2017 Gootenberg et al., 2017 ; Chen et al., 2018 Chen et al., 2018 ; Harrington et al., 2018 Harrington et al., 2018 ; Li et al., 2018a Li et al., 2018a ). More importantly, it has the advantages of high sensitivity, specificity and speed, and low cost ( Bhattacharyya et al., 2018 Bhattacharyya et al., 2018 ). It can be used for the identification of pathogens through nucleic acid detection. The general working principle of the system for nucleic acid detection is as follows: when the Cas protein binds to the target single-/double-stranded DNA (ssDNA/dsDNA) or RNA, the nonspecific ribonuclease activity of the protein is activated, allowing it to specifically cleave the target nucleic acid, nonspecifically cleave adjacent ssDNA or single-stranded RNA (ssRNA), and mark the adjacent nucleic acid with a signal. Signal changes caused by cleavage can be detected with an appropriate detection device to determine whether the tested samples contain the pathogen of interest ( Abudayyeh et al., 2016 Abudayyeh et al., 2016 ; East-Seletsky et al., 2016 East-Seletsky et al., 2016 ; Gootenberg et al., 2017 Gootenberg et al., 2017 , 2018 2018 ; Bhattacharyya et al., 2018 Bhattacharyya et al., 2018 ; Chen et al., 2018 Chen et al., 2018 ; Harrington et al., 2018 Harrington et al., 2018 ; Li et al., 2018b Li et al., 2018b ; Karvelis et al., 2020 Karvelis et al., 2020 ). CRISPR technology can be applied to detect pathogen nucleic acids. The process involves three steps: amplification of the target nucleic acid to amplify the detection signal and improve detection sensitivity; specific recognition of the nucleic acid sequence to activate nonspecific cleavage of adjacent reporter molecules by the Cas protein; and labeling of adjacent nucleic acids with signals, detection of those signals, and interpretation of the results. In this review, we first discuss the discovery and development of the CRISPR-Cas system, briefly summarize the current methods for evaluating the results obtained using the system, and describe in detail its application for the detection of several pathogens that cause infectious diseases, such as human papillomavirus, Zika virus (ZIKV), dengue virus (DENV), avian influenza virus, and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The Cas proteins are classified according to the different proteins used in the detection process. Finally, the prospects and challenges of the CRISPR system for the detection of pathogens of infectious diseases in the future are discussed. 2 Discovery and development of the CRISPR-Cas system 1 Ishino et al. (1987) Ishino et al. (1987) discovered a special structure in the 3' region upstream of the Escherichia coli alkaline phosphatase isozyme gene ( iap ). Five repetitive sequences each containing 29 nucleotides appearing every 32 nucleotides were discovered for the first time as interval and partial palindromic repeats. Then, Jansen et al. (2002) Jansen et al. (2002) used the acronym CRISPR in the genome sequencing of other prokaryotes to identify the family characteristics of CRISPR. Following, Haft et al. (2005) Haft et al. (2005) reported that Cas proteins with nucleolytic activity are often found on both sides of CRISPR sequences. Subsequently, Terns and Terns (2011) Terns and Terns (2011) discovered an adaptive immune system, namely, the CRISPR-Cas system, that fights against invasion by bacteriophages and foreign plasmids in bacteria and archaea. The system consists of RNAs and proteins on both ends ( Fig. 1 Fig. 1 a). The system has since been developed continuously and in 2016 was first recognized for its ability to detect nucleic acids ( Fig. 1 Fig. 1 c) ( Pardee et al., 2016 Pardee et al., 2016 ). The CRISPR-Cas system is divided into two classes depending on the type of Cas protein and the design principle of the effector module ( Makarova et al., 2015 Makarova et al., 2015 ; Koonin et al., 2017 Koonin et al., 2017 ). The class Ⅰ system includes types Ⅰ, Ⅲ, and Ⅳ, which are composed of multiple effector proteins and CRISPR, while the class Ⅱ system includes types Ⅱ, Ⅴ, and Ⅵ, which are composed of one large multidomain multifunctional protein and CRISPR ( Mohanraju et al., 2016 Mohanraju et al., 2016 ). At present, systems including the Cas12 and Cas14 proteins, which are type V Cas proteins, and the Cas13 protein, which is a type VI Cas protein, are most widely used for nucleic acid detection. Below, we review the effects of these three Cas proteins on the collateral cleavage of DNA/RNA. 2.1 Cas12 2 Zetsche et al. (2015) Zetsche et al. (2015) discovered Cpf1 endonuclease (Cas12a) in the class II V-type CRISPR-Cas system of Prevotella and Francisella . FnCpf1 can bind to a CRISPR RNA (crRNA) to form the Cpf-crRNA complex. Under the action of the protospacer-adjacent motif (PAM) sequence in the form of 5'-TTN, it binds to targeted dsDNA to cleave targeted nucleic acids with the RNase H fold (RuvC) domain. This produces staggered double-strand breaks and achieves full cleavage of DNA in vivo and in vitro ( Zetsche et al., 2015 Zetsche et al., 2015 ). In addition to detecting the FnCas12a protein, Li et al. (2018a) Li et al. (2018a) also detected Cas12a proteins from nine randomly selected species such as Francisella tularensis , Lachnospiraceae bacterium ND2006, and Helcococcus kunzii ATCC 51366. They found that Cas12a proteins had the ability to cleave ssDNA in cis and in trans ( Li et al., 2018a Li et al., 2018a ). Subsequently, Chen et al. (2018) Chen et al. (2018) found that the binding of the Cas12a-crRNA complex and targeted DNA through crRNA-complementary ssDNA (PAM sequence-dependent) or dsDNA (TTTT-PAM sequence-dependent) pathway can activate nonspecific cleavage of ssDNA by LbCas12a ( Fig. 1 Fig. 1 b). Among three Cas proteins, LbCas12a, AsCas12a, and FnCas12a, which are widely used in nucleic acid detection, LbCas12a had the strongest lateral cleavage activity and the highest fluorescence intensity ( Dai et al., 2019 Dai et al., 2019 ; Nguyen et al., 2020 Nguyen et al., 2020 ). Most recently, Liu et al. (2021) Liu et al. (2021) identified that Lb2Cas12a can be used as a genome editing tool and has greater flexibility in PAM (5-BYYV-3 editing) selection. Furthermore, the class II V-B effector protein C2c1 was shown to be able to recognize the PAM sequence in the form of 5'-TTN. Under the guidance of single-guide RNA (sgRNA) formed by the combin-ation of crRNA and transactivation crRNA (tracrRNA), the RuvC domain cleaves DNA in vivo and in vitro ( Shmakov et al., 2015 Shmakov et al., 2015 ). Then, Teng et al. (2019) Teng et al. (2019) found that Cas12b can recognize and cleave targeted dsDNA/ssDNA, and can also nonspecifically cleave ssDNA. When targeting dsDNA, Cas12b relies on TTC-PAM and TAC-PAM to accomplish collateral cleavage. On the other hand, when targeting ssDNA, Cas12b does not require a PAM sequence for cleavage activity, and its collateral cleavage activity is higher than that of dsDNA. In addition to the common Cas12a and Cas12b proteins, Cas12 proteins of the class Ⅱ V-type CRISPR-Cas system also have the ability to cleave Cas12d, Cas12h, Cas12i, and CasF (Cas12j) in trans. However, their trans -cleavage activity is relatively weak and does not show strong competitiveness in the field ( Yan WX et al., 2019 Yan WX et al., 2019 ; Pausch et al., 2020 Pausch et al., 2020 ; Leung et al., 2022 Leung et al., 2022 ). The smallest Cas12g protein has only about 800 amino acids and must form a ternary complex with the guide RNA and tracrRNA to induce the cleavage of ssRNA. It also cleaves trans -ssDNA and RNA nonspecifically after it has been activated ( Schnell, 2019 Schnell, 2019 ). 2.2 Cas13 2 Cas13, including its subtypes (i.e., Cas13a, Cas13b, Cas13c, and Cas13d), recognizes and binds ssRNA and acts as an RNA detector ( O'Connell, 2019 O'Connell, 2019 ; Yan FC et al., 2019 Yan FC et al., 2019 ). Shmakov et al. (2015) Shmakov et al. (2015) discovered a single effector protein C2c2 (Cas13a) in the putative class II type VI CRISPR-Cas system. C2c2 has two high-level eukaryotic and prokaryotic nucleotide-binding (HEPN) domains, both of which have RNA nucleases ( Anantharaman et al., 2013 Anantharaman et al., 2013 ). Then, Abudayyeh et al. (2016) Abudayyeh et al. (2016) discovered the CRISPR effector protein class II type VI Cas13a. After recognizing the protospacer flanking sequence (PFS) under the guidance of crRNA, its HEPN domain performs cleavage. Researchers also found that Cas13a cleaves not only targeted RNA, but also nontargeted RNA ( Fig. 1 Fig. 1 b) ( East-Seletsky et al., 2016 East-Seletsky et al., 2016 ). After that, Smargon et al. (2017) Smargon et al. (2017) discovered the type VI-B effector Cas13b which can be divided into the B1 and B2 subtypes depending on the presence of additional proteins; B1 has CSX28 (a small protein encoding a transmembrane domain), while B2 has CSX27 (a protein that contains 3‍–‍4 predicted transmembrane domains). Cas13b cleaves targeted RNA and the adjacent ssRNAs with arbitrary sequences. To date, few detection systems based on Cas13c have been reported. The sequence similarity between the Cas13d protein and the Cas13a-c homolog is low, and Cas13d contains an HEPN domain not typical of the Cas13 superfamily. Based on a series of studies on Cas13d from different bacteria, it was found that Cas13d relies on crRNA, but not on the HEPN domain, to cleave the target ( Konermann, 2018 Konermann, 2018 ). It does not require the flanking sequence of the target and can target any RNA sequence ( Yan et al., 2018 Yan et al., 2018 ). 2.3 Cas14 2 Cas14, the smallest RNA-guided nuclease, was found in Diapherotrites, Parvarchaeota, Aigarchaeota, Nanoarchaeota, Nanohaloarchaeota (DPANN), a superphylum of symbiotic archaea ( Harrington et al., 2018 Harrington et al., 2018 ; Hamm et al., 2019 Hamm et al., 2019 ). Like Cas12, Cas14 has a C-terminal RuvC domain, so it is classified as a V-type effector protein ( Harrington et al., 2018 Harrington et al., 2018 ). Sequence analysis revealed that Cas14 can be divided into three subtypes: Cas14a, Cas14b, and Cas14c. Under the action of the sgRNA complex formed by crRNA and tracrRNA, these proteins recognize complementary target ssDNA via the RuvC domain to achieve cleavage. Cas14 can target ssDNA cleavage without the need for PAM sequences, and can recognize and cleave dsDNA under the guidance of PAM sequences ( Jansen et al., 2002 Jansen et al., 2002 ; Karvelis et al., 2020 Karvelis et al., 2020 ) ( Fig. 1 Fig. 1 b). In addition, in the process of cis -cleavage ssDNA/dsDNA, trans -cleavage of ssDNA bound with the fluorescein group and quenched group is produced, and then the cutting activity of Cas14 is reflected by the fluorescence signal. 3 Signal detection platforms 1 As CRISPR-Cas activity cannot be measured directly, signal detection platforms are required for analysis ( Maffert et al., 2017 Maffert et al., 2017 ). Platforms used for detecting CRISPR-Cas activity can be generally divided into four categories depending on the need for precision instruments, test strips, or other special devices ( Fig. 2 Fig. 2 ). 3.1 Fluorophore-quencher (FQ) double labeling 2 Double labeling is achieved by fluorescence resonance energy transfer between fluorescence compounds and their corresponding quenching compounds. When there is no side branch cutting, fluorescence resonance energy transfer can occur if the distance between two groups of molecules is small (up to a certain range). As a result, the quenching group absorbs the fluorescence energy emitted by the fluorescence group, and no fluorescence signal is observed. On the other hand, separation of the two groups of molecules occurs during collateral cleavage, and the fluorescence energy can no longer be quenched, resulting in a fluorescence signal. By combining double labeling with a fluorescence detection system, the fluorescence signal can be detected in real time under a specific excitation wavelength. However, professionally trained personnel are needed to operate the system, and a large real-time fluorescence detector is needed, limiting the application of this system in the real-time detection of infectious diseases. 3.2 Paper-based lateral flow dipstick 2 Sano et al. (1992) Sano et al. (1992) skillfully combined molecular biology with immunochromatography to establish the lateral flow dipstick (LFD) detection method. Its working principle includes: (1) labeling of detected single-/double-stranded nucleotides with a fluorescent group and biotin, and (2) detection of control lines modified by an anti-fluorescent antibody and streptavidin (a ligand that binds biotin). The sample first binds to the fluorescent antibody (primary antibody) on the sample pad (colloidal gold), and the liquid of the test strip flows under capillary force. When there is no collateral cleavage, the reporter system remains intact and combines only with the secondary antibody on the control line, that is, a band is observed only on the control line. When nonspecific cleavage occurs, the reporter system is divided into the biotin part, which can combine with the detection line, and the antibody part, which can combine with the control line. Thus, two bands are displayed. The number of bands on the test strip can be observed visually. This demonstrates that paper-based lateral flow analysis (LFA) can also be developed as a specific and qualitative detection technology. In addition, paper-based LFA technology can be applied in resource-poor areas due to its portability and the ability to easily visualize the results. 3.3 Direct visual detection with the naked eye 2 Compared to the fluorescence/quenching group and LFD detection methods, direct visual detection does not rely on fluorescence labeling and detection equipment or paper. Direct visual detection is simple and intuitive and allows real-time detection. At present, there are many direct visual detection methods, including the turbidimetric method and detection based on gold nanoparticles (AuNPs) ( Liu et al., 2022 Liu et al., 2022 ). Turbidity refers to the degree of hindrance to light transmission caused by suspended matter in a liquid. The turbidity of a liquid depends on the content of the suspended matter and its characteristics, including size, shape, and refractive index. Visual detection of turbidity is based on the principle of liquid-liquid phase separation, which is the unique phenomenon in which two solutions with different specific gravities and immiscibilities are mixed. The turbidity of a solution can be increased by liquid-liquid phase separation, and this change can be observed with the naked eye ( Hyman et al., 2014 Hyman et al., 2014 ; Aumiller et al., 2016 Aumiller et al., 2016 ). In addition, AuNPs, which have a simple structure, are affordable, do not require special equipment, and can be used to label ss/dsDNA or RNA. When collateral cleavage occurs, the state transformation of nanoparticles (dispersion to aggregation) causes the solution to change color from red to blue-purple, and to appear colorless after centrifugation. A change in the color of the solution indicates the presence of the target pathogen ( Zhang et al., 2021 Zhang et al., 2021 ). 3.4 Other platforms 2 In addition to the above detection methods, methods for on-site detection and diagnosis of nucleic acids include electrochemical method-based biosensors. The detection principle of electrochemical methods includes the triggering of electron transfer on the electrode after collateral cleavage, leading to electrochemical conversion. Changes in signal recorded on a sensor indicate the presence of pathogens ( Dai and Liu, 2019 Dai and Liu, 2019 ; Dai et al., 2019 Dai et al., 2019 ). As microfluidic chips used in microfluidic detection are simple, small, easy to carry, and inexpensive, they can be integrated with microarray technologies for pathogen detection and analysis. Therefore, they have been widely used for real-time detection. In many classifications of microfluidic technologies, a droplet-based microfluidic system has been applied to pathogen analysis due to its advantages of high specific surface area, high flux, no cross contamination, and diverse operation modes ( Iwai, 2022 Iwai, 2022 ). Notably, graphene is a promising photodetector material with an ultra-wide wavelength range (from ultraviolet to terahertz). Its good biocompatibility also facilitates its wide application in the field of nano-electronic biosensors. When CRISPR transistors use inactivated Cas proteins to search for specific targets in biological samples, if the target exists, the charge generated by the biological interaction will be sensed and signaled by graphene. This method can realize detection in the natural state of DNA without amplification or expensive optical instruments ( Hajian et al., 2019 Hajian et al., 2019 ). Furthermore, smartphones have been developed as telemedicine devices, for example for the analysis of pictures of colorimetric test results, and apps can be developed for the quantification of test results ( Shinoda et al., 2021 Shinoda et al., 2021 ). 4 Application of nucleic acid detection of pathogens 1 The CRISPR-Cas system has been widely used to detect viruses and other pathogens, such as bacteria, fungi, and parasites. Table 1 Table 1 summarizes the applications of Cas proteins. CRISPR: clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated protein; HOLMES: one-hour low-cost multipurpose highly efficient system; DETECTR: DNA endonuclease-targeted CRISPR trans reporter; E-CRISPR: electrochemical CRISPR; OR-DETECTR: one-tube detection platform based on reverse transcriptase and recombinase polymerase isothermal amplification (RT-RPA) and DETECTR technology; CDetection: Cas12b-mediated DNA detection; Cas12aVDet: Cas12a-based visual detection; Cas12a-UPTLFA: CRISPR-Cas12 with upconverting phosphor technology (UPT)‍-based lateral flow analysis (LFA); RAA: recombinase-aided amplification; BCA: biological bar code immunoassay; TB-QUICK: Mycobacterium tuberculosis DNA detection platform; SHERLOCK: specific high-sensitivity enzymatic reporter unlocking; HUDSON: heating unextracted diagnostic samples to obliterate nucleases; SHINE: streamlined highlighting of infections to navigate epidemics; CCB: a bacterial detection method based on CRISPR-Cas13a; APC-Cas: a allosteric probe-initiated catalysis and CRISPR-Cas13a system; CMP: Cas14a1-mediated nucleic acid detection platform; ACasB: aptamer Cas14a1 biosensor; PRV: pseudorabies virus; JEV: Japanese encephalitis virus; PB-19: parvovirus B19; SARS-CoV-2: severe acute respiratory syndrome-coronavirus-2; ORF1a: open reading frame 1a; HERC2 : HECT domain and RCC1-like domain 2; PCR: polymerase chain reaction; RT: reverse transcription; LAMP: loop-mediated isothermal amplification; CFU: colony forming unit; FQ: fluorophore-quencher; FAM: carboxyfluorescein; AuNPs: gold nanoparticles; UV-Vis: ultraviolet-visible; MB: methylene blue. CRISPR-Cas system Detection method Pathogens detected Signal amplification method Limit of detection Sensitivity(%) Specificity(%) Requirement for multiplexing Portability Temperature(℃) Time Result determination method Reference 13 CRISPR-Cas12 HOLMES PRV, JEV PCR+CRISPR-Cas12a 1–10 amol/L No No 37 1 h FQ reporter+fluorescence detector Li et al., 2018a Li et al., 2018a DETECTR HPV RPA+CRISPR-Cas12a 1–10 amol/L No No 37 2 h FQ reporter+fluorescence detector Chen et al., 2018 Chen et al., 2018 E-CRISPR HPV16, PB-19 CRISPR-Cas12a 50 pmol/L Yes Yes 0.5 h MB tag+electrochemicalbiosensors Dai et al., 2019 Dai et al., 2019 DETECTR SARS-CoV-2 E/N RT-LAMP+CRISPR-Cas12a 40–60 amol/L 95 100 No No 37–62 <40 min FAM-biotin reporter+lateral flow strips Broughtonet al., 2020 Broughtonet al., 2020 OR-DETECTR SARS-CoV-2 N/RdRp RT-RPA+CRISPR-Cas12a 5 amol/L 100 100 No No 42 50 min FAM-biotin reporter+lateral flow strips Sun et al., 2021 Sun et al., 2021 CDetection HPV RPA+CRISPR-Cas12b 1 amol/L No No 37 FQ reporter+fluorescence plate reader Teng et al., 2019 Teng et al., 2019 Cas12aVDet Mycoplasma RPA+CRISPR-Cas12a 10 amol/L 100 100 No No 37 30 min FQ reporter+naked eye under blue light Wang et al., 2019 Wang et al., 2019 Cas12a-UPTLFA Yersinia pestis RPA+CRISPR-Cas12a 3 amol/L 93.75 90.63 No Yes 37 1.3 h FQ reporter+portable UPT biosensor You et al., 2021 You et al., 2021 CRISPR-Cas12a-powered dual-mode biosensor Salmonella PCR+CRISPR-Cas12a 1 CFU/mL No No 94, 55, 72, and 37 1 h Color changes in AuNPs+naked eye or a portablecolorimeter Ma L et al., 2021 Ma L et al., 2021 RAA-Cas12a-Tg Toxoplasma gondii oocysts RAA+CRISPR-Cas12a 1 fmol/L No No 39 and 37 70 min FQ reporter+microplate reader Ma QN et al., 2021 Ma QN et al., 2021 BCA-RPA-Cas12a Salmonella typhimurium RPA+CRISPR-Cas12a 1 CFU/mL No No 37 <60 min Barcode AuNPs+naked eye under blue light Cai et al., 2021 Cai et al., 2021 RAA-based E-CRISPR Listeria monocytogenes RAA+CRISPR-Cas12a 0.68 amol/L No No 37 80 min FQ reporter+real-time fluorescencequantitative PCR instrument Li et al., 2021b Li et al., 2021b TB-QUICK Mycobacterium tuberculosis LAMP+CRISPR-Cas12b 1.3 copies/μL 86.8 95.2 No No 65 and 48 70–90 min FQ reporter+real-time fluorescencequantitative PCR instrument Sam et al., 2021 Sam et al., 2021 7 CRISPR-Cas13 SHERLOCK Zika virus, Dengue virus RPA+CRISPR-Cas13a 2 amol/L No No 95 and 37 2 h FQ reporter+real-time detection Gootenberget al., 2017 Gootenberget al., 2017 SHERLOCKv2 Zika virus, Dengue virus RPA+CRISPR-Cas13b 8×10 -3 amol/L Yes Yes 37 1.5 h FAM-biotin reporter+lateral flow strips Gootenberget al., 2018 Gootenberget al., 2018 SHERLOCK-HUDSON Zika virus RPA+CRISPR-Cas13 20–90 amol/L 100 100 No Yes 37–50 and 60–95 <2 h FAM-biotin reporter+lateral flow strips Myhrvold et al., 2018 Myhrvold et al., 2018 Porcine reproductive and respiratory syndrome virus RPA+CRISPR-Cas13 172 copies/μL 100 100 No Yes 37 2.5 h FAM-biotin reporter+lateral flow strips Chang et al., 2020 Chang et al., 2020 SHINE SARS-CoV-2 ORF1a RT-RPA+CRISPR-Cas13a 20 amol/L 90 100 No Yes 41 50 min Fluorescence+smartphone app Arizti-Sanzet al., 2020 Arizti-Sanzet al., 2020 CCB Staphylococcus aureus PCR+CRISPR-Cas13a 1 amol/L No No 37 4 h FQ reporter+UV-Vis spectrophotometer Zhou et al., 2020 Zhou et al., 2020 APC-Cas Salmonella enteritidis Conventional real-time PCR+CRISPR-Cas13a 1 CFU/mL 98 100 No No 37 1.5 h FQ reporter+real-time PCR detection systems Shen et al., 2020 Shen et al., 2020 3 CRISPR-Cas14 Cas14-DETECTR Human HERC2 gene PCR+CRISPR-Cas13a No Yes 37 >2 h FQ reporter+fluorescenceplate reader Harrington et al., 2018 Harrington et al., 2018 CMP Eberthella typhi Asymmetric PCR+CRISPR-Cas14a1 1×10 4 amol/L 100 100 No No 51–53 and 37 2 h FQ reporter+fluorescenceplate reader Ge et al., 2021 Ge et al., 2021 ACasB S . aureus Aptamer+CRISPR-Cas14a1 400 CFU/mL No Yes 37 >2 h FQ reporter+fluorescenceplate reader Wei et al., 2022 Wei et al., 2022 4.1 Application of the CRISPR-Cas12 system 2 The combination of Cas12a and Cas12b is the most widely used for the detection of nucleic acids of pathogens via the CRISPR-Cas system. Taking advantage of the collateral cleavage ability of Cas12a, Li et al. (2018a) Li et al. (2018a) used Cas12a to develop the HOLMES (one-hour low-cost multipurpose highly efficient system) technology. They used fluorescence-quenching reporter-labeled ssDNA for the detection of pseudorabies virus and Japanese encephalitis virus with a sensitivity of 1‍‒‍10 amol/L. Chen et al. (2018) Chen et al. (2018) combined recombinase polymerase isothermal amplification (RPA) with LbCas12a detection to establish the DNA endonuclease-targeted CRISPR trans reporter (DETECTR) system, which can specifically detect human papillomavirus (HPV) in cell culture and anal swabs. This method has good sensitivity, as it can accurately identify clinical samples of HPV16/18 typing with differences as small as six base pairs (bp) between genotypes, and shows a good correlation with PCR results. Tsou et al. (2019) Tsou et al. (2019) proposed a CRISPR-Cas12 point-of-care (POC) detection system read by LFD that can directly detect HPV DNA in circulating plasma. The proposed protocol is as follows: (1) the cell supernatant is first treated with lysis buffer, and isothermal amplification is carried out directly without separating the DNA; (2) the amplified sample is then incubated with the CRISPR-Cas12a system; and (3) HPV16 and HPV18 can be detected by LFD with a detection limit consistent with that of PCR (0.24 fmol/L) in 2 h and 55 min. Dai et al. (2019) Dai et al. (2019) proposed an electrochemical biosensor electrochemical CRISPR (E-CRISPR) based on CRISPR-Cas12a. The biosensor uses a methylene blue-labeled nonspecific ssDNA reporter for the detection of HPV16 and parvovirus B19 (PB-‍19) and has a detection limit as low as 50 pmol/L of viral nucleic acids. Subsequently, Xu et al. (2020) Xu et al. (2020) proposed a biosensor platform that combines electrochemical DNA (E-DNA) with CRISPR-Cas12a for the detection of PB-19 ssDNA. This method detects the conformational change of targeted nucleic acids as a result of surface signal probing by electrochemical sensors. The proposed platform can detect viral DNA levels as low as 10 fmol/L without using amplification. Compared with traditional electrochemical detection methods, it has superior sensitivity and specificity. Wang et al. (2020a) Wang et al. (2020a) integrated CRISPR-Cas12a with LFD to establish an African swine fever virus (ASFV) detection method. CRISPR-Cas12a-LFD can detect 20 copies/reaction of ASFV DNA within 1 h without cross-reaction in the presence of other porcine DNA viruses. In addition, real-time quantitative PCR (qPCR) was demonstrated to detect 149 clinical samples simultaneously with a coincidence rate of 100%. He et al. (2020) He et al. (2020) detected pathogen nucleic acids within 2 h at a detection limit as low as 1 pmol/L without amplification of ASFV DNA by combining the CRISPR-Cas12a system with a fluorescence-based POC system. They found that the ternary complex formed by crRNA-Cas12a-ASFV DNA was stable even after 24 h of incubation. Based on this finding, the detection limit may be as low as 100 fmol/L. Wu et al. (2020) Wu et al. (2020) proposed a new method for the detection of ASFV based on CRISPR-Cas12a. The target nucleic acids are first amplified by PCR and subjected to Cas12 detection. Detection is then performed with a probe-based lateral flow biosensor (LFB). This method can be used to detect seven types of ASFV simultaneously at a sensitivity as low as 2.5 fmol/L within 2 h. As the coronavirus disease-2019 (COVID-19) pandemic is ongoing, the CRISPR-Cas12 system has been used to detect the novel coronavirus. Broughton et al. (2020) Broughton et al. (2020) proposed the DETECTR method, which is based on the CRISPR-Cas12 system and incorporates reverse transcription (RT)‍-loop-mediated isothermal amplification (LAMP) and LFD. This method can be used to detect SARS-CoV-2 in 40 min with 95% positive consistency and 100% negative consistency. Guo et al. (2020) Guo et al. (2020) then introduced an integrated SARS-CoV-2 nucleic acid detection platform, CRISPR-assisted detection (CASdetec), which simultaneously combines sample processing, nucleic acid amplification, and Cas12b detection. This system can achieve a detection limit of 1×10 4 copies/mL by connecting the system to a portable blue cassette. Wang et al. (2020c) Wang et al. (2020c) developed a rapid and sensitive visual method for detecting SARS-CoV-2 known as CRISPR-Cas12a-based detection with naked eye readout (NER). It can be used to detect at least ten copies of viral genes in 45 min. Joung et al. (2020) Joung et al. (2020) introduced the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) testing in one pot (STOP) method, which simplifies nucleic acid extraction by using the magnetic bead method and shortens the detection time by incorporating a lateral flow system. LAMP and CRISPR-Cas12b were used to build the platform. The system can be used to detect SARS-CoV-2 within 1 h with 93.1% sensitivity and 98.5% specificity. Ding et al. (2020) Ding et al. (2020) introduced two CRISPR RNAs with no PAM site restriction to improve the detection sensitivity for pathogens, and named this system all-in-one dual (AIOD)‍-CRISPR. AIOD-CRISPR can be used to visually detect SARS-CoV-2 and human immunodeficiency virus (HIV). Ali et al. (2020) Ali et al. (2020) proposed the in vitro specific CRISPR-based assay for nucleic acid detection (iSCAN) system for the detection of SARS-CoV-2. The thermophilic variants of Cas12b and RT-LAMP are used in combination with the dipstick technique to achieve timely detection of SARS-CoV-2. Sun et al. (2021) Sun et al. (2021) proposed a one-tube detection platform based on RT-RPA and DETECTR (OR-DETECTR) and based on RT-RPA and SHERLOCK (OR-SHERLOCK). The detection process takes 50 min, has a detection limit of 1 copy/µL, and does not require sample transfer. Recently, Wang R et al. (2021) Wang R et al. (2021) established a visual SARS-CoV-2 detection system, known as one-pot visual RT-LAMP-CRISPR (opvCRISPR), through the combination of RT-LAMP and Cas12a. Observation of a single molecule by the naked eye can be achieved in 45 min. Subsequently, Ma et al. (2022) Ma et al. (2022) established a visual biosensor based on CRISPR-Cas12a and RT-PCR. In the presence of SARS-CoV-2 virus nucleic acids, the polymerization or dispersion state of AuNPs is affected by the linker DNA. A color change is triggered by a change in the state of AuNPs and can be visualized by the naked eye or a smartphone via a color detection app. This method can be used to successfully detect SARS-CoV-2 genes in synthetic vectors, transcriptional RNA, and SARS-CoV-2 pseudoviruses. It is highly sensitive and specific for the detection of pseudoviruses, has a detection limit of as low as 1 copy/µL, and is not affected by cross-reactivity. CRISPR-Cas12a has also been used for the detection of nonviral pathogens. Cas12a-based visual detection (Cas12aVDet) was established by integrating RPA and Cas12a. Through naked eye observation of the green fluorescence signal emitted by the reporter under blue-light irradiation, DNA can be detected at a single molecular level within 30 min ( Wang et al., 2019 Wang et al., 2019 ). It was demonstrated that this method can successfully detect mycoplasma contamination at a detection rate of up to 100%. Ma L et al. (2021) Ma L et al. (2021) developed a dual-mode biosensor powered by CRISPR-Cas12a. Within the biosensor, Cas12a first identifies target nucleic acids. The target nucleic acid is then amplified by the Salmonella invasion protein A ( invA ) sequence, and linker DNA is cut to disperse aggregated AuNPs. This causes a red to blue-purple color change that can be observed visually. When recorded by a portable colorimeter, pathogenic Salmonella -infected food can be detected at a limit of 1 CFU/mL (CFU: colony forming unit). When it is used in combination with a thermal imager to explore the photothermal effect of AuNPs, the detection limit is also 1 CFU/mL, and the dynamic range is 1‍-‍1×10 8 CFU/mL. Ma QN et al. (2021) Ma QN et al. (2021) also combined recombinant enzyme-assisted amplification (RAA) with the CRISPR-Cas12a system to introduce the RAA-Cas12a-Tg detection system. Targeting the 529-bp repeat element ( RE ) gene and internal transcribed spacer-1 ( ITS-1 ) gene, the platform shows strong specificity for Toxoplasma gondii oocysts in soil samples. The platform also has a lower limit of detection (1 fmol/L) for the 529-bp RE gene of T. gondii , showing better detection sensitivity than conventional PCR. Cai et al. (2021) Cai et al. (2021) combined biological bar code immunoassay (BCA), RPA, and CRISPR-Cas12a to construct a triple signal amplification system (BCA-RPA-Cas12a) for quick detection of Salmonella typhimurium in milk within 1 h. Target bacteria are first separated by immunomagnetic nanoparticles and marked with an AuNP probe for signal amplification. The bio-barcode DNA is amplified isothermally by RPA, and then trans -cleavage of activated CRISPR-Cas12a occurs. The detection of pathogens can then be performed visually via the generated fluorescence signal under blue light. Li et al. (2021b) Li et al. (2021b) established the E-CRISPR based on RAA. After nucleic acid-targeted cleavage, the number of surface signal probes containing electrochemical labels changes, generating an electrochemical signal difference that indicates the existence of a targeted pathogen. This method was demonstrated to detect 0.64 amol/L DNA of Listeria monocytogenes (135 CFU/mL in pure culture). It can also detect Flammulina velutipes . You et al. (2021) You et al. (2021) integrated CRISPR-Cas12 with upconverting phosphor technology (UPT)‍-based LFA (Cas12a-UPTLFA) for the detection of Yersinia pestis . Y . pestis DNA in blood can be detected with high sensitivity (93.75%) and high specificity (90.63%). To avoid aerosol contamination caused by transferring amplification products, Li et al. (2021a) Li et al. (2021a) developed a Cas12aFDet platform based on fluorescence detection that can amplify the production of L. monocytogenes -targeted nucleic acids and enhance Cas12a detection by PCR or RAA within 15 min in an airtight reaction tube. The results showed that the detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet for L . monocytogenes serotype 4c are 33.7 CFU/mL and 135.0 CFU/mL, respectively. Furthermore, Cas12aFDet based on RAA can be used to specifically detect L . monocytogenes DNA at a detection limit as low as 0.64 amol/L. Li et al. (2019) Li et al. (2019) used thermophilic RNA-guided Cas12b, which has DNA endonuclease activity, and asymmetric PCR amplification or LAMP to create the HOLMESv2 platform. The platform can be used to detect 1×10 -8 nmol/L DNA virus in urine samples. The integrated amplification and detection steps can be optimized in a single reaction system to reduce cross-contamination. When combined with Bst3.0 DNA polymerase, Japanese encephalitis virus RNA can be detected within 1 h. Teng et al. (2019) Teng et al. (2019) established a Cas12b-based DNA detection platform, namely Cas12b-mediated DNA detection (CDetection), using AaCas12b-sgRNA and dsDNA activators to identify HPV16 and HPV18. The experimental results showed that 1 amol/L HPV DNA in human plasma can be detected when the platform is used in combination with RPA. HPV DNA can be detected rapidly with high resolution when the tuned guide RNA (tgRNA) used for CDetection is optimized. Sam et al. (2021) Sam et al. (2021) integrated LAMP and CRISPR-Cas12b to create a novel Mycobacterium tuberculosis ( M . tb ) DNA detection platform (TB-QUICK). The sputum and bronchoalveolar lavage fluid (BALF) of 138 tuberculosis patients, 21 nontuberculosis patients, and 61 people in close contact with tuberculosis were analyzed, using the M . tb IS6110 gene as the target gene. The detection limit was 1.3 copies/μL, and there was no cross-reaction with other strains. The method could effectively distinguish tuberculous from non-tuberculous samples. This method had higher sensitivity (86.8%) and specificity (95.2%) than conventional detection methods using acid-fast bacillus (AFB) smears, i.e., M . tb culture and GeneXpert M. tuberculosis and rifampin (MTB/RIF; Xpert) assays. Huang et al. (2021) Huang et al. (2021) developed a platform for the rapid detection of Campylobacter jejuni within 40 min based on CRISPR-Cas12b. About 10 CFU/mL of C . jejuni contamination could be detected in chicken samples with a sensitivity 10 times higher than that of the traditional method. 4.2 Application of the CRISPR-Cas13 system 2 East-Seletsky et al. (2016) East-Seletsky et al. (2016) demonstrated for the first time that the LbuCas13a protein can be used to detect target RNA. However, detection sensitivity was unsatisfactory and could not meet the requirement for clinical diagnosis. Gootenberg et al. (2017) Gootenberg et al. (2017) proposed SHERLOCK, which combines RPA, T7 RNA polymerase, and Cas13a to detect the target RNA. The system was demonstrated to specifically detect 2 amol/L ZIKV and DENV RNAs. The researchers further developed SHERLOCKv2, which involves four Cas proteins (i.‍e., LwaCas13a, PsmCas13b, CcaCas13b, and AsCas12a) and four fluorescent groups (i.‍e., carboxyfluorescein (FAM), Texas, cyanine 5, and hexachlorofluorescein), to qualitatively detect four different types of nucleic acids (i.‍e., synthetic ssRNA, dsDNA, ZIKV ssRNA, and DENV ssRNA). SHERLOCKv2 can achieve single molecule detection with a limit as low as 2 amol/L within 2 h when RPA is combined with LFD ( Gootenberg et al., 2018 Gootenberg et al., 2018 ). Myhrvold et al. (2018) Myhrvold et al. (2018) then proposed the SHERLOCK heating unextracted diagnostic samples to obliterate nucleases (HUDSON) platform, which successfully omits the nucleic acid extraction step for the detection of pathogens. This method can detect as few as 1 copy/µL ZIKV and DENV RNA within 2 h when combined with LDF. Chang et al. (2020) Chang et al. (2020) successfully combined RPA, T7 reverse transcriptase, and CRISPR-Cas13a to generate a system to detect porcine reproductive and respiratory syndrome virus (PRRSV) at 37 ℃. This enhanced Cas13a detection method can be used for real-time analysis or intuitive reading and can detect the virus specifically and sensitively at a molecular level as low as 172 copies/µL. Liu et al. (2019) Liu et al. (2019) designed specific crRNAs targeting in vitro transcribed RNA of the hemagglutinin (HA) and neuraminidase (NA) genes of the H7N9 virus. They combined this system with the RT-RPA technique and achieved a minimum detection limit of 1 fmol/L HA and NA genes in 50 min without cross-reaction in the presence of other subtypes of viruses. Wu et al. (2019) Wu et al. (2019) screened for and detected nasopharyngeal carcinoma and other Epstein-Barr virus (EBV)‍-related diseases at room temperature by optimizing the functions of amplification primers in the RPA step of SHERLOCK. Through analysis of 48 nasopharyngeal carcinoma samples and 50 negative control plasma samples, the system achieved a 96% detection rate of positive samples and detected no negative samples, showing good agreement with qPCR results. Qin et al. (2019) Qin et al. (2019) used Cas13-integrated microfluidic technology to analyze Ebola virus RNA. Using a customized fluorimeter, purified virus RNA samples of 20 PFU/mL can be detected in 5 min. Most strikingly, Ackerman et al. (2020) Ackerman et al. (2020) established an extended multipathogen detection platform, namely combinatorial arrayed reactions for multiplexed evaluation of nucleic acids (CARMEN), for multiple nucleic acid evaluation in one step by integrating Cas13 with a microwell array. This system can be used to stably detect 4500 crRNA targets on a single array and distinguish 169 human-associated viruses. Recently, Wang S et al. (2021) Wang S et al. (2021) established PCR-CRISPR technology with a minimum detection limit of 1 copy/reaction hepatitis B virus (HBV) DNA in 15 min. qPCR was able to detect HBV DNA in only 302 of 312 serum samples, while PCR-CRISPR detected HBV DNA in all the samples. The use of CRISPR-Cas13 for SARS-CoV-2 detection has been widely reported. A test strip for the detection of SARS-CoV-2 was developed in March 2020 by targeting the S and ORF1ab genes of the virus using SHERLOCK. The test strip can detect targeted nucleic acid sequences in the range of 20‍‒200 amol/L ( Zhang et al., 2020 Zhang et al., 2020 ). The SHERLOCK TM CRISPR SARS-CoV-2 kit has been authorized for emergency use by the United States Food and Drug Administration (FDA). Hou et al. (2020) Hou et al. (2020) proposed a CRISPR-COVID method for detecting SARS-CoV-2 RNA by combining RPA and CRISPR-Cas13a. The method can be used to detect SARS-CoV-2 in 40 min and has a sensitivity close to a single copy. The streamlined highlighting of infections to navigate epidemics (SHINE) platform established by Arizti-Sanz et al. (2020) Arizti-Sanz et al. (2020) can be used to detect SARS-CoV-2 RNA in unextracted samples. Read-out is achieved via fluorescence detection. The sensitivity and specificity of 50 nasopharyngeal swabs were 90% and 100%, respectively. Fozouni et al. (2021) Fozouni et al. (2021) developed a CRISPR-Cas13a analysis method for detecting viral RNA in nasopharyngeal swab samples through a mobile phone. The analysis can be performed without amplification with a detection limit of 100 copies/µL in 30 min. Shinoda et al. (2021) Shinoda et al. (2021) also established a CRISPR-based amplification-free digital RNA detection (SATORI) method. This method combines CRISPR-Cas13 detection with microchamber array technology to achieve nonamplified RNA detection. It allows detection of 10 fmol/L ssRNA in 5 min. The detection sensitivity of the system can be further improved by other crRNA methods to allow detection of 5 fmol/L SARS-CoV-2 RNA. Liu et al. (2022) Liu et al. (2022) established an AuNPs-labeled CRISPR-Cas13a detection system using solid-state nanopore sensing technology, which showed great sensitivity and specificity for viral RNA sensing. When CRISPR is activated, AuNPs are released from the non-covalent coupling of AuNPs and RNA, and a strong ionic signal pulse from the nanopore sensor is detected to identify the SARS-CoV-2 target RNA. In the absence of front-end target amplification, 50 fmol/L SARS-CoV-2 RNA could be detected at room temperature, which is close to the sensitivity required for rapid screening of SARS-CoV-2. Shen et al. (2020) Shen et al. (2020) developed an allosteric probe-initiated catalysis and CRISPR-Cas13a (APC-Cas) system. The platform uses nucleic acid aptamers to construct AP DNA molecules, which are transformed into their active configurations after binding to the targeted pathogens. After AP is transformed into an active configuration, the primer is annealed to the binding site region. Combined with isothermal amplification and CRISPR-Cas13a detection, the detection platform can be used to detect a single copy of Streptococcus enteritis DNA without pathogen isolation and nucleic acid extraction. With a dynamic range of 1×10 5 CFU/mL, the system is more sensitive than qPCR. Zhou et al. (2020) Zhou et al. (2020) established a bacterial detection method based on CRISPR-Cas13a (CCB). When combined with PCR, this method can be used to detect as little as 1 amol/L Staphylococcus aureus genomic DNA with a detection limit of 1 CFU/mL within 4 h. Compared with traditional bacterial culture methods, CCB is highly sensitive and rapid. 4.3 Application of the CRISPR-Cas14 system 2 The CRISPR system based on Cas14 can be used not only for single-nucleotide polymorphism (SNP) typing, but also for nucleic acid detection. Harrington et al. (2018) Harrington et al. (2018) established a high-fidelity SNP genotyping method with Cas14-DETECTR, which was successfully applied to HECT domain and RCC1-like domain 2 ( HERC2 ) gene typing of human eye color (blue and brown) differentiation. Aquino-Jarquin (2019) Aquino-Jarquin (2019) used HUDSON for rapid detection of small and nonenveloped human Boca virus ssDNA in nasopharyngeal swabs and sediment of respiratory infections in children. More recently, Ge et al. (2021) Ge et al. (2021) used Cas14a1 without the restriction of PAM loci. It has a strong recognition ability for ssDNA and can cleave ssDNA nonspecifically. Cas14a1 was successfully integrated to generate a Cas14a1-mediated nucleic acid detection platform (CMP) combined with asymmetric PCR for the detection of Streptococcus pyogenes and Eberthella typhi ( Ge et al., 2021 Ge et al., 2021 ). Cleavage from Cas14a1 can be analyzed via a microplate reader. The method has proved effective in identifying seven pathogenic bacteria. It has good reproducibility and strong specificity in identifying bacteria without cross-reaction, and has great potential for pathogen detection and identification. Recently, Wei et al. (2022) Wei et al. (2022) developed a detection platform based on an aptamer Cas14a1 biosensor (ACasB). Its working principle is roughly as follows: first, the specific aptamers of S . aureus are hybridized with the blocker DNA. After the addition of live S . aureus , the blocker can be released during bacteria-aptamer binding. The released blocker can eventually activate Cas14a1 protein by binding to sgRNA, resulting in a change in fluorescence intensity. This method can achieve high specificity and sensitivity for live S . aureus without extraction or amplification of nucleic acid. Experiments have proved that this method can directly detect live S . aureus cells from 400 CFU/mL. Compared with qPCR, the Cas14a1 aptamer biosensor can accurately detect S . aureus in complex samples, demonstrating its potential for application in food safety and environmental monitoring ( Wei et al., 2022 Wei et al., 2022 ). 5 Conclusions and future perspectives 1 Infectious diseases, such as COVID-19, pose great threats to the survival of human beings. It is important to develop accurate and rapid technologies for detecting pathogens, the culprits of infectious diseases. Compared with existing detection methods, CRISPR technology is rapid and simple, and has low cost, high sensitivity, and high specificity. Use of the CRISPR-Cas system can accelerate the detection of trace nucleic acids of pathogens. RT-PCR has satisfactory sensitivity and specificity in the detection of low target concentrations and has been recognized as the gold standard for the detection of most pathogens. However, the ability of crRNA to recognize complementary target sequences based on Cas proteins makes the CRISPR-Cas system more specific than primer-based detection systems for the detection of pathogens. Nevertheless, more accurate bioinformatics tools that allow the design of more specific and active crRNAs are needed to overcome the poor reliability of crRNA design. Among the reported CRISPR detection methods, CRISPR-Cas12-based detection techniques, including HOLMES, DETECTR, and CDetection, have been used to detect pseudorabies virus, different genotypes of HPV, SARS-CoV-2, and other pathogens. The abilities of CRISPR-Cas13-based detection techniques, including SHERLOCK, SHERLOCKv2, and CRISPR-COVID, to detect ZIKV, DENV, and SARS-CoV-2, have also been studied. CRISPR-Cas14-based detection techniques, including Cas14a1, have been used to detect bacteria in milk samples. The properties of different Cas proteins may vary depending on their sources. For example, the concentration of Mn 2+ does not necessarily increase the sensitivity of all Cas12a proteins. Therefore, the improvement and modification of Cas enzymes in the future are important for further optimization of their functions. Although CRISPR-Cas technology is easy to use and does not require special equipment, it requires additional mechanisms for analysis and interpretation. Detecting the results requires signal detection platforms such as double-labeled fluorescence quenching reporters, LFD, naked eye visibility, electrochemistry, or microfluidic chips. Analysis and interpretation of results obtained using the CRISPR-Cas system may increase the complexity of the system to a certain extent. However, methods based on LFD or visual observation are straightforward and broaden the application of CRISPR-Cas technology from the detection of pathogens to on-the-spot diagnosis of pathogens. Although methods based on electrochemistry or microfluidic chips still rely heavily on sophisticated instruments, these methods also show great advantages in the high-throughput detection of multiple pathogens. For example, CARMEN is being used to achieve this goal. While it would be highly desirable to integrate the four parts of the CRISPR detection system, namely sample processing, pre-amplification, CRISPR detection, and result reading, in a single device for use in a wider scope of applications and commercialization, it remains challenging to incorporate all the parts at present. Many attempts have been made to refine each part of CRISPR: (1) simplification of sample preparation or the introduction of a one-pot reaction system to shorten the sample preparation time; (2) rapid nucleic acid extraction without amplification; (3) optimization of Cas enzymes and crRNA for sensitivity and specificity enhancement of detection; (4) design of multi-channel or high-throughput detection systems to reduce operating costs; and (5) the adoption of a portable reading mode for detection, an amplification-free strategy, and/or improved amplification technology to simplify the system. As current research on CRISPR-Cas-based testing systems is in full swing, we believe that this technology will shine in the foreseeable future. References Abudayyeh OO , Gootenberg JS , Konermann S , et al. , 2016 . C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector . Science , 353 ( 6299 ): aaf5573 . https://doi.org/10.1126/science.aaf5573 https://doi.org/10.1126/science.aaf5573 C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector Ackerman CM , Myhrvold C , Thakku SG , et al. , 2020 . Massively multiplexed nucleic acid detection with Cas13 . Nature , 582 ( 7811 ): 277 - 282 . https://doi.org/10.1038/s41586-020-2279-8 https://doi.org/10.1038/s41586-020-2279-8 Massively multiplexed nucleic acid detection with Cas13 Ali Z , Aman R , Mahas A , et al. , 2020 . iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2 . Virus Res , 288 : 198129 . https://doi.org/10.1016/j.virusres.2020.198129 https://doi.org/10.1016/j.virusres.2020.198129 iSCAN: an RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2 Anantharaman V , Makarova KS , Burroughs AM , et al. , 2013 . Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing . Biol Direct , 8 : 15 . https://doi.org/10.1186/1745-6150-8-15 https://doi.org/10.1186/1745-6150-8-15 Comprehensive analysis of the HEPN superfamily: identification of novel roles in intra-genomic conflicts, defense, pathogenesis and RNA processing Aquino-Jarquin G , 2019 . CRISPR-Cas14 is now part of the artillery for gene editing and molecular diagnostic . Nanomedicine , 18 : 428 - 431 . https://doi.org/10.1016/j.nano.2019.03.006 https://doi.org/10.1016/j.nano.2019.03.006 CRISPR-Cas14 is now part of the artillery for gene editing and molecular diagnostic Arizti-Sanz J , Freije CA , Stanton AC , et al. , 2020 . Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2 . Nat Commun , 11 : 5921 . https://doi.org/10.1038/s41467-020-19097-x https://doi.org/10.1038/s41467-020-19097-x Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2 Aumiller WM , Cakmak FP , Davis BW , et al. , 2016 . RNA-based coacervates as a model for membraneless organelles: formation, properties, and interfacial liposome assembly . Langmuir , 32 ( 39 ): 10042 - 10053 . https://doi.org/10.1021/acs.langmuir.6b02499 https://doi.org/10.1021/acs.langmuir.6b02499 RNA-based coacervates as a model for membraneless organelles: formation, properties, and interfacial liposome assembly Bhattacharyya RP , Thakku SG , Hung DT , 2018 . Harnessing CRISPR effectors for infectious disease diagnostics . ACS Infect Dis , 4 ( 9 ): 1278 - 1282 . https://doi.org/10.1021/acsinfecdis.8b00170 https://doi.org/10.1021/acsinfecdis.8b00170 Harnessing CRISPR effectors for infectious disease diagnostics Broughton JP , Deng XD , Yu GX , et al. , 2020 . CRISPR-Cas12-based detection of SARS-CoV-2 . Nat Biotechnol , 38 ( 7 ): 870 - 874 . https://doi.org/10.1038/s41587-020-0513-4 https://doi.org/10.1038/s41587-020-0513-4 CRISPR-Cas12-based detection of SARS-CoV-2 Cai QQ , Wang R , Qiao ZH , et al. , 2021 . Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system . Analyst , 146 ( 17 ): 5271 - 5279 . https://doi.org/10.1039/D1AN00717C https://doi.org/10.1039/D1AN00717C Single-digit Salmonella detection with the naked eye using bio-barcode immunoassay coupled with recombinase polymerase amplification and a CRISPR-Cas12a system Chang YF , Deng Y , Li TY , et al. , 2020 . Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a . Transbound Emerg Dis , 67 ( 2 ): 564 - 571 . https://doi.org/10.1111/tbed.13368 https://doi.org/10.1111/tbed.13368 Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a Chen JS , Ma EB , Harrington LB , et al. , 2018 . CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity . Science , 360 ( 6387 ): 436 - 439 . https://doi.org/10.1126/science.aar6245 https://doi.org/10.1126/science.aar6245 CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity Dai YF , Liu CC , 2019 . Recent advances on electrochemical biosensing strategies toward universal point-of-care systems . Angew Chem Int Ed Engl , 131 ( 36 ): 12483 - 12496 . https://doi.org/10.1002/ange.201901879 https://doi.org/10.1002/ange.201901879 Recent advances on electrochemical biosensing strategies toward universal point-of-care systems Dai YF , Somoza RA , Wang L , et al. , 2019 . Exploring the trans-cleavage activity of CRISPR-Cas12a (cpf1) for the development of a universal electrochemical biosensor . Angew Chem Int Ed Engl , 131 ( 48 ): 17560 - 17566 . https://doi.org/10.1002/ange.201910772 https://doi.org/10.1002/ange.201910772 Exploring the trans-cleavage activity of CRISPR-Cas12a (cpf1) for the development of a universal electrochemical biosensor Ding X , Yin K , Li ZY , et al. , 2020 . Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay . Nat Commun , 11 : 4711 . https://doi.org/10.1038/s41467-020-18575-6 https://doi.org/10.1038/s41467-020-18575-6 Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay East-Seletsky A , O'Connell MR , Knight SC , et al. , 2016 . Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection . Nature , 538 ( 7624 ): 270 - 273 . https://doi.org/10.1038/nature19802 https://doi.org/10.1038/nature19802 Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection Fozouni P , Son S , de León Derby MD , et al. , 2021 . Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy . Cell , 184 ( 2 ): 323 - 333.e9 . https://doi.org/10.1016/j.cell.2020.12.001 https://doi.org/10.1016/j.cell.2020.12.001 Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy Ge XL , Meng T , Tan X , et al. , 2021 . Cas14a1-mediated nucleic acid detectifon platform for pathogens . Biosens Bioelectron , 189 : 113350 . https://doi.org/10.1016/j.bios.2021.113350 https://doi.org/10.1016/j.bios.2021.113350 Cas14a1-mediated nucleic acid detectifon platform for pathogens Gootenberg JS , Abudayyeh OO , Lee JW , et al. , 2017 . Nucleic acid detection with CRISPR-Cas13a/C2c2 . Science , 356 ( 6336 ): 438 - 442 . https://doi.org/10.1126/science.aam9321 https://doi.org/10.1126/science.aam9321 Nucleic acid detection with CRISPR-Cas13a/C2c2 Gootenberg JS , Abudayyeh OO , Kellner MJ , et al. , 2018 . Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 . Science , 360 ( 6387 ): 439 - 444 . https://doi.org/10.1126/science.aaq0179 https://doi.org/10.1126/science.aaq0179 Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6 Guo L , Sun XH , Wang XE , et al. , 2020 . SARS-CoV-2 detection with CRISPR diagnostics . Cell Discov , 6 : 34 . https://doi.org/10.1038/s41421-020-0174-y https://doi.org/10.1038/s41421-020-0174-y SARS-CoV-2 detection with CRISPR diagnostics Haft DH , Selengut J , Mongodin EF , et al. , 2005 . A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes . PLoS Comput Biol , 1 ( 6 ): e60 . https://doi.org/10.1371/journal.pcbi.0010060 https://doi.org/10.1371/journal.pcbi.0010060 A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes Hajian R , Balderston S , Tran T , et al. , 2019 . Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor . Nat Biomed Eng , 3 ( 6 ): 427 - 437 . https://doi.org/10.1038/s41551-019-0371-x https://doi.org/10.1038/s41551-019-0371-x Detection of unamplified target genes via CRISPR-Cas9 immobilized on a graphene field-effect transistor Hamm JN , Erdmann S , Eloe-Fadrosh EA , et al. , 2019 . Unexpected host dependency of Antarctic Nanohaloarchaeota . Proc Natl Acad Sci USA , 116 ( 29 ): 14661 - 14670 . https://doi.org/10.1073/pnas.1905179116 https://doi.org/10.1073/pnas.1905179116 Unexpected host dependency of Antarctic Nanohaloarchaeota Harrington LB , Burstein D , Chen JS , et al. , 2018 . Programmed DNA destruction by miniature CRISPR-Cas14 enzymes . Science , 362 ( 6416 ): 839 - 842 . https://doi.org/10.1126/science.aav4294 https://doi.org/10.1126/science.aav4294 Programmed DNA destruction by miniature CRISPR-Cas14 enzymes He Q , Yu DM , Bao MD , et al. , 2020 . High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system . Biosens Bioelectron , 154 : 112068 . https://doi.org/10.1016/j.bios.2020.112068 https://doi.org/10.1016/j.bios.2020.112068 High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system Hou TY , Zeng WQ , Yang ML , et al. , 2020 . Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19 . PLoS Pathog , 16 ( 8 ): e1008705 . https://doi.org/10.1371/journal.ppat.1008705 https://doi.org/10.1371/journal.ppat.1008705 Development and evaluation of a rapid CRISPR-based diagnostic for COVID-19 Huang Y , Gu D , Xue H , et al. , 2021 . Rapid and accurate Campylobacter jejuni detection with CRISPR-Cas12b based on newly identified Campylobacter jejuni -specific and -conserved genomic signatures . Front Microbiol , 12 : 649010 . https://doi.org/10.3389/fmicb.2021.649010 https://doi.org/10.3389/fmicb.2021.649010 Rapid and accurate Campylobacterjejuni detection with CRISPR-Cas12b based on newly identified Campylobacterjejuni-specific and -conserved genomic signatures Hwang H , Hwang BY , Bueno J , 2018 . Biomarkers in infectious diseases . Dis Markers , 2018 : 8509127 . https://doi.org/10.1155/2018/8509127 https://doi.org/10.1155/2018/8509127 Biomarkers in infectious diseases Hyman AA , Weber CA , Jülicher F , 2014 . Liquid-liquid phase separation in biology . Ann Rev Cell Dev Biol , 30 : 39 - 58 . https://doi.org/10.1146/annurev-cellbio-100913-013325 https://doi.org/10.1146/annurev-cellbio-100913-013325 Liquid-liquid phase separation in biology Ishino Y , Shinagawa H , Makino K , et al. , 1987 . Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli , and identification of the gene product . J Bacteriol , 169 ( 12 ): 5429 - 5433 . https://doi.org/10.1128/jb.169.12.5429-5433.1987 https://doi.org/10.1128/jb.169.12.5429-5433.1987 Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product Iwai K , Wwhrs M , Garber M , et al. , 2022 . Scalable and automated CRISPR-based strain engineering using droplet microfluidics . Microsyst Nanoeng , 8 : 31 . https://doi.org/10.1038/s41378-022-00357-3 https://doi.org/10.1038/s41378-022-00357-3 Scalable and automated CRISPR-based strain engineering using droplet microfluidics Jansen R , van Embden JDA , Gaastra W , et al. , 2002 . Identification of genes that are associated with DNA repeats in prokaryotes . Mol Microbiol , 43 ( 6 ):‍ 1565 - 1575 . https://doi.org/10.1046/j.1365-2958.2002.02839.x https://doi.org/10.1046/j.1365-2958.2002.02839.x Identification of genes that are associated with DNA repeats in prokaryotes Joung J , Ladha A , Saito M , et al. , 2020 . Detection of SARS-CoV-2 with SHERLOCK one-pot testing . N Engl J Med , 383 ( 15 ): 1492 - 1494 . https://doi.org/10.1056/NEJMc2026172 https://doi.org/10.1056/NEJMc2026172 Detection of SARS-CoV-2 with SHERLOCK one-pot testing Karvelis T , Bigelyte G , Young JK , et al. , 2020 . PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage . Nucleic Acids Res , 48 ( 9 ): 5016 - 5023 . https://doi.org/10.1093/nar/gkaa208 https://doi.org/10.1093/nar/gkaa208 PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage Konermann S , Lotfy P , Brideau NJ , et al. , 2018 . Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors . Cell , 173 ( 3 ): 665 - 676.e14 . https://doi.org/10.1016/j.cell.2018.02.033 https://doi.org/10.1016/j.cell.2018.02.033 Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors Koonin EV , Makarova KS , Zhang F , 2017 . Diversity, classification and evolution of CRISPR-Cas systems . Curr Opin Microbiol , 37 : 67 - 78 . https://doi.org/10.1016/j.mib.2017.05.008 https://doi.org/10.1016/j.mib.2017.05.008 Diversity, classification and evolution of CRISPR-Cas systems Kostyusheva A , Brezgin S , Babin Y , et al. , 2022 . CRISPR-Cas systems for diagnosing infectious diseases . Methods , 203 : 431 - 446 . https://doi.org/10.1016/j.ymeth.2021.04.007 https://doi.org/10.1016/j.ymeth.2021.04.007 CRISPR-Cas systems for diagnosing infectious diseases Leung RKK , Cheng QX , Wu ZL , et al. , 2022 . CRISPR-Cas12-based nucleic acids detection systems . Methods , 203 : 276 - 281 . https://doi.org/10.1016/j.ymeth.2021.02.018 https://doi.org/10.1016/j.ymeth.2021.02.018 CRISPR-Cas12-based nucleic acids detection systems Li F , Ye QH , Chen MT , et al. , 2021a . Cas12aFDet: a CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeria monocytogenes serotype 4c . Anal Chim Acta , 1151 : 338248 . https://doi.org/10.1016/j.aca.2021.338248 https://doi.org/10.1016/j.aca.2021.338248 Cas12aFDet: a CRISPR/Cas12a-based fluorescence platform for sensitive and specific detection of Listeriamonocytogenes serotype 4c Li F , Ye QH , Chen MT , et al. , 2021b . An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeria monocytogenes detection . Biosens Bioelectron , 179 :‍ 113073 . https://doi.org/10.1016/j.bios.2021.113073 https://doi.org/10.1016/j.bios.2021.113073 An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for Listeriamonocytogenes detection Li LX , Li SY , Wu N , et al. , 2019 . HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation . ACS Synth Biol , 8 ( 10 ): 2228 - 2237 . https://doi.org/10.1021/acssynbio.9b00209 https://doi.org/10.1021/acssynbio.9b00209 HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation Li SY , Cheng QX , Wang JM , et al. , 2018a . CRISPR-Cas12a-assisted nucleic acid detection . Cell Discov , 4 : 20 . https://doi.org/10.1038/s41421-018-0028-z https://doi.org/10.1038/s41421-018-0028-z CRISPR-Cas12a-assisted nucleic acid detection Li SY , Cheng QX , Liu JK , et al. , 2018b . CRISPR-Cas12a has both cis - and trans -cleavage activities on single-stranded DNA . Cell Res , 28 ( 4 ): 491 - 493 . https://doi.org/10.1038/s41422-018-0022-x https://doi.org/10.1038/s41422-018-0022-x CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA Liu L , Xu ZH , Awayda K , et al. , 2022 . Gold nanoparticle-labeled CRISPR-Cas13a assay for the sensitive solid-state nanopore molecular counting . Adv Mater Technol , 7 ( 3 ): 2101550 . https://doi.org/10.1002/admt.202101550 https://doi.org/10.1002/admt.202101550 Gold nanoparticle-labeled CRISPR-Cas13a assay for the sensitive solid-state nanopore molecular counting Liu XY , Lin L , Tang LC , et al. , 2021 . Lb2Cas12a and its engin Lb2Cas12a and its engin eered variants mediate genome editing in human cells . FASEB J , 35 ( 4 ): e21270 . https://doi.org/10.1096/fj.202001013RR https://doi.org/10.1096/fj.202001013RR eered variants mediate genome editing in human cells Liu YF , Xu HP , Liu C , et al. , 2019 . CRISPR-Cas13a nanomachine based simple technology for avian influenza a (H7N9) virus on-site detection . J Biomed Nanotechnol , 15 ( 4 ): 790 - 798 . https://doi.org/10.1166/jbn.2019.2742 https://doi.org/10.1166/jbn.2019.2742 CRISPR-Cas13a nanomachine based simple technology for avian influenza a (H7N9) virus on-site detection Lozano R , Naghavi M , Foreman K , et al. , 2012 . Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010 . Lancet , 380 ( 9859 ): 2095 - 2128 . https://doi.org/10.1016/S0140-6736(12)61728-0 https://doi.org/10.1016/S0140-6736(12)61728-0 Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010 Ma L , Peng L , Yin LJ , et al. , 2021 . CRISPR-Cas12a-powered dual-mode biosensor for ultrasensitive and cross-validating detection of pathogenic bacteria . ACS Sens , 6 ( 8 ): 2920 - 2927 . https://doi.org/10.1021/acssensors.1c00686 https://doi.org/10.1021/acssensors.1c00686 CRISPR-Cas12a-powered dual-mode biosensor for ultrasensitive and cross-validating detection of pathogenic bacteria Ma L , Yin LJ , Li XY , et al. , 2022 . A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics . Biosens Bioelectron , 195 : 113646 . https://doi.org/10.1016/j.bios.2021.113646 https://doi.org/10.1016/j.bios.2021.113646 A smartphone-based visual biosensor for CRISPR-Cas powered SARS-CoV-2 diagnostics Ma QN , Wang M , Zheng LB , et al. , 2021 . RAA-Cas12a-Tg: a nucleic acid detection system for Toxoplasma gondii based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) . Microorganisms , 9 ( 8 ): 1644 . https://doi.org/10.3390/microorganisms9081644 https://doi.org/10.3390/microorganisms9081644 RAA-Cas12a-Tg: a nucleic acid detection system for Toxoplasmagondii based on CRISPR-Cas12a combined with recombinase-aided amplification (RAA) Maffert P , Reverchon S , Nasser W , et al. , 2017 . New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings . Eur J Clin Microbiol Infect Dis , 36 ( 10 ): 1717 - 1731 . https://doi.org/10.1007/s10096-017-3013-9 https://doi.org/10.1007/s10096-017-3013-9 New nucleic acid testing devices to diagnose infectious diseases in resource-limited settings Makarova KS , Wolf YI , Alkhnbashi OS , et al. , 2015 . An updated evolutionary classification of CRISPR-Cas systems . Nat Rev Microbiol , 13 ( 11 ): 722 - 736 . https://doi.org/10.1038/nrmicro3569 https://doi.org/10.1038/nrmicro3569 An updated evolutionary classification of CRISPR-Cas systems Matthijs G , Souche E , Alders M , et al. , 2016 . Guidelines for diagnostic next-generation sequencing . Eur J Hum Genet , 24 ( 1 ): 2 - 5 . https://doi.org/10.1038/ejhg.2015.226 https://doi.org/10.1038/ejhg.2015.226 Guidelines for diagnostic next-generation sequencing Mohanraju P , Makarova KS , Zetsche B , et al. , 2016 . Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems . Science , 353 ( 6299 ): aad5147 . https://doi.org/10.1126/science.aad5147 https://doi.org/10.1126/science.aad5147 Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems Myhrvold C , Freije CA , Gootenberg JS , et al. , 2018 . Field-deployable viral diagnostics using CRISPR-Cas13 . Science , 360 ( 6387 ): 444 - 448 . https://doi.org/10.1126/science.aas8836 https://doi.org/10.1126/science.aas8836 Field-deployable viral diagnostics using CRISPR-Cas13 Nguyen LT , Smith BM , Jain PK , 2020 . Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection . Nat Commun , 11 : 4906 . https://doi.org/10.1038/s41467-020-18615-1 https://doi.org/10.1038/s41467-020-18615-1 Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection O'Connell MR , 2019 . Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR-Cas systems . J Mol Biol , 431 ( 1 ): 66 - 87 . https://doi.org/10.1016/j.jmb.2018.06.029 https://doi.org/10.1016/j.jmb.2018.06.029 Molecular mechanisms of RNA targeting by Cas13-containing type VI CRISPR-Cas systems Pardee K , Green AA , Takahashi MK , et al. , 2016 . Rapid, low-cost detection of Zika virus using programmable biomolecular components . Cell , 165 ( 5 ): 1255 - 1266 . https://doi.org/10.1016/j.cell.2016.04.059 https://doi.org/10.1016/j.cell.2016.04.059 Rapid, low-cost detection of Zika virus using programmable biomolecular components Pausch P , Al-Shayeb B , Bisom-Rapp E , et al. , 2020 . CRISPR-CasΦ from huge phages is a hypercompact genome editor . Science , 369 ( 6501 ): 333 - 337 . https://doi.org/10.1126/science.abb1400 https://doi.org/10.1126/science.abb1400 CRISPR-CasΦ from huge phages is a hypercompact genome editor Qin PW , Park M , Alfson KJ , et al. , 2019 . Rapid and fully microfluidic ebola virus detection with CRISPR-Cas13a . ACS Sens , 4 ( 4 ): 1048 - 1054 . https://doi.org/10.1021/acssensors.9b00239 https://doi.org/10.1021/acssensors.9b00239 Rapid and fully microfluidic ebola virus detection with CRISPR-Cas13a Sam IK , Chen YY , Ma J , et al. , 2021 . TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis . J Infect , 83 ( 1 ): 54 - 60 . https://doi.org/10.1016/j.jinf.2021.04.032 https://doi.org/10.1016/j.jinf.2021.04.032 TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacteriumtuberculosis Sano T , Smith CL , Cantor CR , 1992 . Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates . Science , 258 ( 5079 ): 120 - 122 . https://doi.org/10.1126/science.1439758 https://doi.org/10.1126/science.1439758 Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates Scheler O , Glynn B , Kurg A , 2014 . Nucleic acid detection technologies and marker molecules in bacterial diagnostics . Expert Rev Mol Diagn , 14 ( 4 ): 489 - 500 . https://doi.org/10.1586/14737159.2014.908710 https://doi.org/10.1586/14737159.2014.908710 Nucleic acid detection technologies and marker molecules in bacterial diagnostics Schnell C , 2019 . Quantum imaging in biological samples . Nat Methods , 16 ( 3 ): 214 . https://doi.org/10.1038/s41592-019-0346-6 https://doi.org/10.1038/s41592-019-0346-6 Quantum imaging in biological samples Shen JJ , Zhou XM , Shan YY , et al. , 2020 . Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction . Nat Commun , 11 : 267 . https://doi.org/10.1038/s41467-019-14135-9 https://doi.org/10.1038/s41467-019-14135-9 Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction Shinoda H , Taguchi Y , Nakagawa R , et al. , 2021 . Amplification-free RNA detection with CRISPR-Cas13 . Commun Biol , 4 : 476 . https://doi.org/10.1038/s42003-021-02001-8 https://doi.org/10.1038/s42003-021-02001-8 Amplification-free RNA detection with CRISPR-Cas13 Shmakov S , Abudayyeh OO , Makarova KS , et al. , 2015 . Discovery and functional characterization of diverse class 2 CRISPR-Cas systems . Mol Cell , 60 ( 3 ): 385 - 397 . https://doi.org/10.1016/j.molcel.2015.10.008 https://doi.org/10.1016/j.molcel.2015.10.008 Discovery and functional characterization of diverse class 2 CRISPR-Cas systems Smargon AA , Cox DBT , Pyzocha NK , et al. , 2017 . Cas13b is a type VI-B CRISPR-associated RNA-guided rnase differentially regulated by accessory proteins Csx27 and Csx28 . Mol Cell , 65 ( 4 ): 618 - 630.e7 . https://doi.org/10.1016/j.molcel.2016.12.023 https://doi.org/10.1016/j.molcel.2016.12.023 Cas13b is a type VI-B CRISPR-associated RNA-guided rnase differentially regulated by accessory proteins Csx27 and Csx28 Sun YY , Yu L , Liu CX , et al. , 2021 . One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a . J Transl Med , 19 : 74 . https://doi.org/10.1186/s12967-021-02741-5 https://doi.org/10.1186/s12967-021-02741-5 One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a Taylor SC , Laperriere G , Germain H , 2017 . Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data . Sci Rep , 7 : 2409 . https://doi.org/10.1038/s41598-017-02217-x https://doi.org/10.1038/s41598-017-02217-x Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data Teng F , Guo L , Cui TT , et al. , 2019 . CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity . Genome Biol , 20 : 132 . https://doi.org/10.1186/s13059-019-1742-z https://doi.org/10.1186/s13059-019-1742-z CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity Terns MP , Terns RM , 2011 . CRISPR-based adaptive immune systems . Curr Opin Microbiol , 14 ( 3 ): 321 - 327 . https://doi.org/10.1016/j.mib.2011.03.005 https://doi.org/10.1016/j.mib.2011.03.005 CRISPR-based adaptive immune systems Tsou JH , Leng QX , Jiang F , 2019 . A CRISPR test for detection of circulating nuclei acids . Transl Oncol , 12 ( 12 ): 1566 - 1573 . https://doi.org/10.1016/j.tranon.2019.08.011 https://doi.org/10.1016/j.tranon.2019.08.011 A CRISPR test for detection of circulating nuclei acids Wang B , Wang R , Wang DQ , et al. , 2019 . Cas12aVDet: a CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection . Anal Chem , 91 ( 19 ): 12156 - 12161 . https://doi.org/10.1021/acs.analchem.9b01526 https://doi.org/10.1021/acs.analchem.9b01526 Cas12aVDet: a CRISPR/Cas12a-based platform for rapid and visual nucleic acid detection Wang R , Qian CY , Pang YN , et al. , 2021 . opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection . Biosens Bioelectron , 172 : 112766 . https://doi.org/10.1016/j.bios.2020.112766 https://doi.org/10.1016/j.bios.2020.112766 opvCRISPR: one-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection Wang S , Li H , Kou Z , et al. , 2021 . Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system . Clin Microbiol Infect , 27 ( 3 ): 443 - 450 . https://doi.org/10.1016/j.cmi.2020.04.018 https://doi.org/10.1016/j.cmi.2020.04.018 Highly sensitive and specific detection of hepatitis B virus DNA and drug resistance mutations utilizing the PCR-based CRISPR-Cas13a system Wang XJ , Ji PP , Fan HY , et al. , 2020a . CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus . Commun Biol , 3 : 62 . https://doi.org/10.1038/s42003-020-0796-5 https://doi.org/10.1038/s42003-020-0796-5 CRISPR/Cas12a technology combined with immunochromatographic strips for portable detection of African swine fever virus Wang XJ , Shang XY , Huang XX , 2020b . Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods . Emerg Microbes Infect , 9 ( 1 ): 1682 - 1691 . https://doi.org/10.1080/22221751.2020.1793689 https://doi.org/10.1080/22221751.2020.1793689 Next-generation pathogen diagnosis with CRISPR/Cas-based detection methods Wang XJ , Zhong MT , Liu Y , et al. , 2020c . Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER . Sci Bull , 65 ( 17 ): 1436 - 1439 . https://doi.org/10.1016/j.scib.2020.04.041 https://doi.org/10.1016/j.scib.2020.04.041 Rapid and sensitive detection of COVID-19 using CRISPR/Cas12a-based detection with naked eye readout, CRISPR/Cas12a-NER Wei YD , Tao ZZ , Wan L , et al. , 2022 . Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection . Biosens Bioelectron , 211 : 114282 . https://doi.org/10.1016/j.bios.2022.114282 https://doi.org/10.1016/j.bios.2022.114282 Aptamer-based Cas14a1 biosensor for amplification-free live pathogenic detection Wu JH , Mukama O , Wu W , et al. , 2020 . A CRISPR/Cas12a based universal lateral flow biosensor for the sensitive and specific detection of African swine-fever viruses in whole blood . Biosensors , 10 ( 12 ): 203 . https://doi.org/10.3390/bios10120203 https://doi.org/10.3390/bios10120203 A CRISPR/Cas12a based universal lateral flow biosensor for the sensitive and specific detection of African swine-fever viruses in whole blood Wu YT , Liu SX , Wang F , et al. , 2019 . Room temperature detection of plasma Epstein-Barr virus DNA with CRISPR-Cas13 . Clin Chem , 65 ( 4 ): 591 - 592 . https://doi.org/10.1373/clinchem.2018.299347 https://doi.org/10.1373/clinchem.2018.299347 Room temperature detection of plasma Epstein-Barr virus DNA with CRISPR-Cas13 Xu W , Jin T , Dai YF , et al. , 2020 . Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems . Biosens Bioelectron , 155 : 112100 . https://doi.org/10.1016/j.bios.2020.112100 https://doi.org/10.1016/j.bios.2020.112100 Surpassing the detection limit and accuracy of the electrochemical DNA sensor through the application of CRISPR Cas systems Yan FC , Wang W , Zhang JQ , 2019 . CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR-Cas9 . Cell Biol Toxicol , 35 ( 6 ): 489 - 492 . https://doi.org/10.1007/s10565-019-09489-1 https://doi.org/10.1007/s10565-019-09489-1 CRISPR-Cas12 and Cas13: the lesser known siblings of CRISPR-Cas9 Yan WX , Chong SR , Zhang HB , et al. , 2018 . Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein . Mol Cell , 70 ( 2 ): 327 - 339.e5 . https://doi.org/10.1016/j.molcel.2018.02.028 https://doi.org/10.1016/j.molcel.2018.02.028 Cas13d is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein Yan WX , Hunnewell P , Alfonse LE , et al. , 2019 . Functionally diverse type V CRISPR-Cas systems . Science , 363 ( 6422 ): 88 - 91 . https://doi.org/10.1126/science.aav7271 https://doi.org/10.1126/science.aav7271 Functionally diverse type V CRISPR-Cas systems You Y , Zhang PP , Wu GS , et al. , 2021 . Highly specific and sensitive detection of Yersinia pestis by portable Cas12a-UPTLFA platform . Front Microbiol , 12 : 700016 . https://doi.org/10.3389/fmicb.2021.700016 https://doi.org/10.3389/fmicb.2021.700016 Highly specific and sensitive detection of Yersiniapestis by portable Cas12a-UPTLFA platform Yu ACH , Vatcher G , Yue X , et al. , 2012 . Nucleic acid-based diagnostics for infectious diseases in public health affairs . Front Med , 6 ( 2 ): 173 - 186 . https://doi.org/10.1007/s11684-012-0195-5 https://doi.org/10.1007/s11684-012-0195-5 Nucleic acid-based diagnostics for infectious diseases in public health affairs Zetsche B , Gootenberg JS , Abudayyeh OO , et al. , 2015 . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system . Cell , 163 ( 3 ): 759 - 771 . https://doi.org/10.1016/j.cell.2015.09.038 https://doi.org/10.1016/j.cell.2015.09.038 Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system Zhang F , Abudayyeh OO , Gootenberg JS , 2020 . A protocol for detection of COVID-19 using CRISPR diagnostics . https://broad.io/sherlockprotocol https://broad.io/sherlockprotocol A protocol for detection of COVID-19 using CRISPR diagnostics Zhang YQ , Chen MY , Liu CR , et al. , 2021 . Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP . Sens Actuators B Chem , 345 : 130411 . https://doi.org/10.1016/j.snb.2021.130411 https://doi.org/10.1016/j.snb.2021.130411 Sensitive and rapid on-site detection of SARS-CoV-2 using a gold nanoparticle-based high-throughput platform coupled with CRISPR/Cas12-assisted RT-LAMP Zhao YX , Chen F , Li Q , et al. , 2015 . Isothermal amplification of nucleic acids . Chem Rev , 115 ( 22 ):‍ 12491 - 12545 . https://doi.org/10.1021/acs.chemrev.5b00428 https://doi.org/10.1021/acs.chemrev.5b00428 Isothermal amplification of nucleic acids Zhou J , Yin LJ , Dong YN , et al. , 2020 . CRISPR-Cas13a based bacterial detection platform: sensing pathogen Staphylococcus aureus in food samples . Anal Chim Acta , 1127 : 225 - 233 . https://doi.org/10.1016/j.aca.2020.06.041 https://doi.org/10.1016/j.aca.2020.06.041 CRISPR-Cas13a based bacterial detection platform: sensing pathogen Staphylococcusaureus in food samples Hongdan GAO prepared the manuscript of the article, including all figures and tables. Zifang SHANG completed the literature collection, the article writing, and the drawing of all the figures and tables. Siew Yin CHAN provided the proofreading of the article. Dongli MA provided guidance and suggestions for the article writing. All authors have read and approved the final version. This work was supported by the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2020JQ-148), the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515110793), and the Shenzhen Pathogen High-Throughput Sequencing Technical Engineering Laboratory Upgrading Project (No. 2019-986), China. 10.1631/jzus.B2200068 1673-1581（2022）11-0881-18 This work was supported by the Natural Science Basic Research Plan in Shaanxi Province of China (No. 2020JQ-148), the Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515110793), and the Shenzhen Pathogen High-Throughput Sequencing Technical Engineering Laboratory Upgrading Project (No. 2019-986), China. 2022-02-10 2022-06-06 2022-11-15 2022-12-27 2022-10-16 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 Hongdan GAO, Zifang SHANG, Siew Yin CHAN, and Dongli MA declare that there are no conflicts of interest. This review 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） 11 23