Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system

doi:10.3785/j.issn.1008-9209.2022.08.051

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (5): 618-632 DOI: 10.3785/j.issn.1008-9209.2022.08.051

Young Scientist Forum

Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system

Hongzhao LI^1,²(

),Hao WANG²(

),Rui YIN²,Min YUE^1,²,Yan LI^1,²

^1.Hainan Institute of Zhejiang University, Sanya 572025, Hainan, China
^2.Institute of Preventive Veterinary Sciences/Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China

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Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (CRISPR/Cas) system, an ancient bacterial and archaeal immune system, has rapidly developed into a popular gene-editing tool, which largely promotes the development of several biology-related fields. By combining the CRISPR/Cas systems with the isothermal amplification techniques, the novel and effective detection methods with high sensitivity and independence of equipment have been established, such as DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). These new technologies not only improve the performance of the CRISPR/Cas system in different situations, but also inspire its application potential in the on-site detection. In this review, we summarized the nucleic acid detection methods developed on the three widely-used CRISPR/Cas systems (CRISPR/Cas9, CRISPR/Cas12a, and CRISPR/Cas13), and elucidated their biological significance and the principles of action. We also reviewed the recent studies on the applications of CRISPR/Cas systems in pathogen detection, and analyzed the characteristics and possible defects of different detection systems in practical applications. This review aims to provide more constructive advice on developing adaptable and efficient CRISPR/Cas-based detection methods for different pathogens in various practical scenarios.

Key words： CRISPR/Cas system Cas protein pathogen nucleic acid detection method

Received: 05 August 2022 Published: 03 November 2023

CLC:

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Corresponding Authors: Yan LI E-mail: 22017116@zju.edu.cn;11917042@zju.edu.cn

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Cite this article:

Hongzhao LI,Hao WANG,Rui YIN,Min YUE,Yan LI. Research advances on pathogenic nucleic acid detection technology based on CRIPSR/Cas system. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(5): 618-632.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.08.051 OR https://www.zjujournals.com/agr/Y2023/V49/I5/618

基于CRISPR/Cas系统的病原核酸检测技术研究进展

作为一种古老的细菌和古菌免疫系统，规律成簇的间隔短回文重复序列及其相关蛋白［clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas), CRISPR/Cas］系统现已发展为新兴的热门基因编辑工具，极大地推动了其他多个生物学相关领域的发展。其中，通过结合CRISPR/Cas系统与核酸恒温扩增技术建立了一些新型的高效、灵敏、不依赖仪器设备的检测方法，如DNA内切酶靶向的CRISPR反式报告系统（DNA endonuclease-targeted CRISPR trans-reporter, DETECTR）、特异性高灵敏度的酶报告器系统（specific high-sensitivity enzymatic reporter unlocking, SHERLOCK）等，不但提高了CRISPR/Cas系统在不同应用场景下的检测性能，更进一步激发了其在现场检测中的应用潜力。本文基于近年来被广泛应用的3种CRISPR/Cas系统（CRISPR/Cas9、CRISPR/Cas12a以及CRISPR/Cas13）的生物核酸检测方法，阐述了CRISPR/Cas系统的生物学意义及一般作用原理，并通过回顾以往开展的CRISPR/Cas系统相关的病原检测研究，比较分析了不同检测系统的特点以及在实际应用过程中可能存在的不足，为针对不同病原在不同应用场景下建立更加高效、合理的CRISPR/Cas系统检测方法提供了有益参考。

关键词： CRISPR/Cas系统, Cas蛋白, 病原, 核酸, 检测方法

Fig. 1 Classification of class 2 CRISPR/Cas systemREC: Recognition lobe; PI: PAM-interacting domain; HEPN: Higher eukaryotes and prokaryotes nucleotide-binding domain; TM: Trans-membrane domain.

Fig. 2 Specific recognition and cleavage of target nucleic acid by Cas9, Cas12a, and Cas13a and non-specific cleavage of single-stranded DNA/RNA by Cas12a and Cas13a

Fig. 3 Amplification principles of RPA and HDA technologies

Fig. 4 Amplification principle of LAMP techniqueA-B. Cyclic template synthesis stages; C. Cyclic amplification and recycling stages.

Table 1 Applications of CRISPR/Cas system in the detection of RNA pathogens

Table 2 Applications of CRISPR/Cas system in the detection of DNA pathogens

Fig. 5 Operating procedures based on CRISPR/Cas12a and CRISPR/Cas13a detection systems


[1]	MARTZY R, KOLM C, KRSKA R, et al. Challenges and perspectives in the application of isothermal DNA amplifi-cation methods for food and water analysis[J]. Analytical and Bioanalytical Chemistry, 2019, 411(9): 1695-1702. DOI: 10.1007/s00216-018-1553-1 doi: 10.1007/s00216-018-1553-1

[2]	RATH D, AMLINGER L, RATH A, et al. The CRISPR-Cas immune system: biology, mechanisms and applications[J]. Biochimie, 2015, 117: 119-128. DOI: 10.1016/j.biochi.2015.03.025 doi: 10.1016/j.biochi.2015.03.025

[3]	MAKAROVA K S, WOLF Y I, KOONIN E V. Comparative genomics of defense systems in archaea and bacteria[J]. Nucleic Acids Research, 2013, 41(8): 4360-4377. DOI: 10.1093/nar/gkt157 doi: 10.1093/nar/gkt157

[4]	GILBERT L A, LARSON M H, MORSUT L, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[J]. Cell, 2013, 154(2): 442-451. DOI: 10.1016/j.cell.2013.06.044 doi: 10.1016/j.cell.2013.06.044

[5]	AGARWAL N, History GUPTA R., evolution and classification of CRISPR-Cas associated systems[J]. Progress in Molecular Biology and Translational Science, 2021, 179: 11-76. DOI: 10.1016/bs.pmbts.2020.12.012 doi: 10.1016/bs.pmbts.2020.12.012

[6]	NAJAH S, SAULNIER C, PERNODET J L, et al. Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci[J]. BMC Biotechnology, 2019, 19: 18. DOI: 10.1186/s12896-019-0509-7 doi: 10.1186/s12896-019-0509-7

[7]	BAO A L, BURRITT D J, CHEN H F, et al. The CRISPR/Cas9 system and its applications in crop genome editing[J]. Critical Reviews in Biotechnology, 2019, 39(3): 321-336. DOI: 10.1080/07388551.2018.1554621 doi: 10.1080/07388551.2018.1554621

[8]	GASIUNAS G, BARRANGOU R, HORVATH P, et al. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria[J]. PNAS, 2012, 109(39): E2579-E2586. DOI: 10.1073/pnas.1208507109 doi: 10.1073/pnas.1208507109

[9]	WATTERS K E, FELLMANN C, BAI H B, et al. Systematic discovery of natural CRISPR-Cas12a inhibitors[J]. Science, 2018, 362(6411): 236-239. DOI: 10.1126/science.aau5138 doi: 10.1126/science.aau5138

[10]	MAKAROVA K S, KOONIN E V. Annotation and Classifi-cation of CRISPR-Cas systems[M]//LUNDGREN M, CHARPENTIER E, FINERAN P C. CRISPR. Methods in Molecular Biology. Vol. 1311. New York: Humana Press, 2015: 47-75. DOI: 10.1007/978-1-4939-2687-9_4 doi: 10.1007/978-1-4939-2687-9_4

[11]	CHEN P, ZHOU J, WAN Y B, et al. A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing[J]. Genome Biology, 2020, 21: 78. DOI: 10.1186/s13059-020-01989-2 doi: 10.1186/s13059-020-01989-2

[12]	ZETSCHE B, GOOTENBERG J S, ABUDAYYEH O O, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system[J]. Cell, 2015, 163(3): 759-771. DOI: 10.1016/j.cell.2015.09.038 doi: 10.1016/j.cell.2015.09.038

[13]	JIANG Y, QIAN F H, YANG J J, et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum [J]. Nature Communications, 2017, 8: 15179. DOI: 10.1038/ncomms15179 doi: 10.1038/ncomms15179

[14]	DONG D, REN K, QIU X L, et al. The crystal structure of Cpf1 in complex with CRISPR RNA[J]. Nature, 2016, 532(7600): 522-526. DOI: 10.1038/nature17944 doi: 10.1038/nature17944

[15]	BANAKAR R, SCHUBERT M, COLLINGWOOD M, et al. Comparison of CRISPR-Cas9/Cas12a ribonucleoprotein complexes for genome editing efficiency in the rice phytoene desaturase (OsPDS)gene[J]. Rice, 2020, 13: 4. DOI: 10.1186/s12284-019-0365-z doi: 10.1186/s12284-019-0365-z

[16]	YAMANO T, NISHIMASU H, ZETSCHE B, et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA[J]. Cell, 2016, 165(4): 949-962. DOI: 10.1016/j.cell.2016.04.003 doi: 10.1016/j.cell.2016.04.003

[17]	MAULE G, CASINI A, MONTAGNA C, et al. Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing[J]. Nature Communications, 2019, 10: 3556. DOI: 10.1038/s41467-019-11454-9 doi: 10.1038/s41467-019-11454-9

[18]	KIM D, KIM J, HUR J K, et al. Erratum: genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells[J]. Nature Biotechnology, 2016, 34(8): 888. DOI: 10.1038/nbt0816-888a doi: 10.1038/nbt0816-888a

[19]	SWARTS D C, JINEK M. Cas9 versus Cas12a/Cpf1: structure-function comparisons and implications for genome editing[J]. Wiley Interdisciplinary Reviews. RNA, 2018, 9(5): e1481. DOI: 10.1002/wrna.1481 doi: 10.1002/wrna.1481

[20]	SHMAKOV S, ABUDAYYEH O O, MAKAROVA K S, et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems[J]. Molecular Cell, 2015, 60(3): 385-397. DOI: 10.1016/j.molcel.2015.10.008 doi: 10.1016/j.molcel.2015.10.008

[21]	LIU L, LI X Y, MA J, et al. The molecular architecture for RNA-guided RNA cleavage by Cas13a[J]. Cell, 2017, 170(4): 714-726.e10. DOI: 10.1016/j.cell.2017.06.050 doi: 10.1016/j.cell.2017.06.050

[22]	GOOTENBERG J S, ABUDAYYEH O O, LEE J W, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2[J]. Science, 2017, 356(6336): 438-442. DOI: 10.1126/science.aam9321 doi: 10.1126/science.aam9321

[23]	KELLNER M J, KOOB J G, GOOTENBERG J S, et al. SHERLOCK: nucleic acid detection with CRISPR nucleases[J]. Nature Protocols, 2019, 14(10): 2986-3012. DOI: 10.1038/s41596-019-0210-2 doi: 10.1038/s41596-019-0210-2

[24]	SMARGON A A, COX D B T, PYZOCHA N K, et al. Cas13b is a type Ⅵ-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28[J]. Molecular Cell, 2017, 65(4): 618-630. DOI: 10.1016/j.molcel.2016.12.023 doi: 10.1016/j.molcel.2016.12.023

[25]	COX D B T, GOOTENBERG J S, ABUDAYYEH O O, et al. RNA editing with CRISPR-Cas13[J]. Science, 2017, 358(6366): 1019-1027. DOI: 10.1126/science.aaq0180 doi: 10.1126/science.aaq0180

[26]	KONERMANN S, LOTFY P, BRIDEAU N J, et al. Trans-criptome engineering with RNA-targeting type Ⅵ-D CRISPR effectors[J]. Cell, 2018, 173(3): 665-676. DOI: 10.1016/j.cell.2018.02.033 doi: 10.1016/j.cell.2018.02.033

[27]	ZHANG B, YE Y M, YE W W, et al. Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d[J]. Nature Communications, 2019, 10: 2544. DOI: 10.1038/s41467-019-10507-3 doi: 10.1038/s41467-019-10507-3

[28]	ZHOU H B, SU J L, HU X D, et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice[J]. Cell, 2020, 181(3): 590-603. DOI: 10.1016/j.cell.2020.03.024 doi: 10.1016/j.cell.2020.03.024

[29]	BROGAN D J, CHAVERRA-RODRIGUEZ D, LIN C P, et al. Development of a rapid and sensitive casrx-based diagnostic assay for SARS-CoV-2[J]. ACS Sensors, 2021, 6(11): 3957-3966. DOI: 10.1021/acssensors.1c01088 doi: 10.1021/acssensors.1c01088

[30]	SAIKI R K, GELFAND D H, STOFFEL S, et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase[J]. Science, 1988, 239(4839): 487-491.

[31]	HEID C A, STEVENS J, LIVAK K J, et al. Real time quantitative PCR[J]. Genome Research, 1996, 6(10): 986-994.

[32]	VOGELSTEIN B, KINZLER K W. Digital PCR[J]. PNAS, 1999, 96(16): 9236-9241.

[33]	WANG Q, ZHANG B B, XU X H, et al. CRISPR-typing PCR (ctPCR), a new Cas9-based DNA detection method[J]. Scientific Reports, 2018, 8: 14126. DOI: 10.1038/s41598-018-32329-x doi: 10.1038/s41598-018-32329-x

[34]	ZHANG B B, WANG Q, XU X H, et al. Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique[J]. Analytical and Bioanalytical Chemistry, 2018, 410(12): 2889-2900. DOI: 10.1007/s00216-018-0873-5 doi: 10.1007/s00216-018-0873-5

[35]	ZHANG B B, XIA Q, WANG Q, et al. Detecting and typing target DNA with a novel CRISPR-typing PCR (ctPCR) technique[J]. Analytical Biochemistry, 2018, 561/562: 37-46. DOI: 10.1016/j.ab.2018.09.012 doi: 10.1016/j.ab.2018.09.012

[36]	GAO J L, WU L, YANG D D, et al. A one-pot CRISPR/Cas9-typing PCR for DNA detection and genotyping[J]. The Journal of Molecular Diagnostics, 2021, 23(1): 46-60. DOI: 10.1016/j.jmoldx.2020.10.004 doi: 10.1016/j.jmoldx.2020.10.004

[37]	PIEPENBURG O, WILLIAMS C H, STEMPLE D L, et al. DNA detection using recombination proteins[J]. PLoS Biology, 2006, 4(7): e204. DOI: 10.1371/journal.pbio.0040204 doi: 10.1371/journal.pbio.0040204

[38]	XU Y, KIM H J, KAYS A, et al. Simultaneous amplification and screening of whole plasmids using the T7 bacteriophage replisome[J]. Nucleic Acids Research, 2006, 34(13): e98. DOI: 10.1093/nar/gkl547 doi: 10.1093/nar/gkl547

[39]	VINCENT M, XU Y, KONG H M. Helicase-dependent isothermal DNA amplification[J]. Scientific Report, 2004, 5(8): 795-800. DOI: 10.1038/sj.embor.7400200 doi: 10.1038/sj.embor.7400200

[40]	GOLDMEYER J, KONG H M, TANG W. Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection[J]. The Journal of Molecular Diagnostics, 2007, 9(5): 639-644. DOI: 10.2353/jmoldx.2007.070012 doi: 10.2353/jmoldx.2007.070012

[41]	NOTOMI T, OKAYAMA H, MASUBUCHI H, et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research, 2000, 28(12): e63. DOI: 10.1093/nar/28.12.e63 doi: 10.1093/nar/28.12.e63

[42]	PARDEE K, GREEN A A, TAKAHASHI M K, et al. Rapid, low-cost detection of Zika virus using programmable bio-molecular components[J]. Cell, 2016, 165(5): 1255-1266. DOI: 10.1016/j.cell.2016.04.059 doi: 10.1016/j.cell.2016.04.059

[43]	ZHOU W H, HU L, YING L M, et al. A CRISPR-Cas9-triggered strand displacement amplification method for ultra-sensitive DNA detection[J]. Nature Communications, 2018, 9: 5012. DOI: 10.1038/s41467-018-07324-5 doi: 10.1038/s41467-018-07324-5

[44]	ZHANG Y H, QIAN L, WEI W J, et al. Paired design of dCas9 as a systematic platform for the detection of featured nucleic acid sequences in pathogenic strains[J]. ACS Synthetic Biology, 2017, 6(2): 211-216. DOI: 10.1021/acssynbio.6b00215 doi: 10.1021/acssynbio.6b00215

[45]	CHEN J S, MA E, HARRINGTON L B, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J]. Science, 2018, 360(6387): 436-439. DOI: 10.1126/science.aar6245 doi: 10.1126/science.aar6245

[46]	LI Y Y, MANSOUR H, WANG T, et al. Naked-eye detection of grapevine red-blotch viral infection using a plasmonic CRISPR Cas12a assay[J]. Analytical Chemistry, 2019, 91(18): 11510-11513. DOI: 10.1021/acs.analchem.9b03545 doi: 10.1021/acs.analchem.9b03545

[47]	LU S H, TONG X H, HAN Y, et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a[J]. Nature Biomedical Engineering, 2022, 6(3): 286-297. DOI: 10.1038/s41551-022-00861-x doi: 10.1038/s41551-022-00861-x

[48]	ABUDAYYEH O O, GOOTENBERG J S, KONERMANN S, et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector[J]. Science, 2016, 353(6299): aaf5573. DOI: 10.1126/science.aaf5573 doi: 10.1126/science.aaf5573

[49]	EAST-SELETSKY A, O’CONNELL M R, KNIGHT S C, et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection[J]. Nature, 2016, 538(7624): 270-273. DOI: 10.1038/nature19802 doi: 10.1038/nature19802

[50]	GOOTENBERG J S, ABUDAYYEH O O, KELLNER M J, et al. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[J]. Science, 2018, 360(6387): 439-444. DOI: 10.1126/science.aaq0179 doi: 10.1126/science.aaq0179

[51]	MYHRVOLD C, FREIJE C A, GOOTENBERG J S, et al. Field-deployable viral diagnostics using CRISPR-Cas13[J]. Science, 2018, 360(6387): 444-448. DOI: 10.1126/science.aas8836 doi: 10.1126/science.aas8836

[52]	ZHANG Y H, WANG Y, XU L Z, et al. Paired dCas9 design as a nucleic acid detection platform for pathogenic strains[J]. Methods, 2022, 203: 70-77. DOI: 10.1016/j.ymeth.2021.06.003 doi: 10.1016/j.ymeth.2021.06.003

[53]	LIU S Y, TAO D G, LIAO Y Y, et al. Highly sensitive CRISPR/Cas12a-based fluorescence detection of porcine reproductive and respiratory syndrome virus[J]. ACS Synthetic Biology, 2021, 10(10): 2499-2507. DOI: 10.1021/acssynbio.1c00103 doi: 10.1021/acssynbio.1c00103

[54]	DING X, YIN K, LI Z Y, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay[J]. Nature Communications, 2020, 11: 4711. DOI: 10.1038/s41467-020-18575-6 doi: 10.1038/s41467-020-18575-6

[55]	ENGLISH M A, SOENKSEN L R, GAYET R V, et al. Programmable CRISPR-responsive smart materials[J]. Science, 2019, 365(6455): 780-785. DOI: 10.1126/science.aaw5122 doi: 10.1126/science.aaw5122

[56]	LIU Y F, XU H P, LIU C, et al. CRISPR-Cas13a nano-machine based simple technology for avian influenza A (H7N9) virus on-site detection[J]. Journal of Biomedical Nanotech-nology, 2019, 15(4): 790-798. DOI: 10.1166/jbn.2019.2742 doi: 10.1166/jbn.2019.2742

[57]	YAO R, XU Y R, WANG L, et al. CRISPR-Cas13a-based detection for bovine viral diarrhea virus[J]. Frontiers in Veterinary Science, 2021, 8: 603919. DOI: 10.3389/fvets.2021.603919 doi: 10.3389/fvets.2021.603919

[58]	ARIZTI-SANZ J, FREIJE C A, STANTON A C, et al. Stream-lined inactivation, amplification, and Cas13-based detection of SARS-CoV-2[J]. Nature Communications, 2020, 11: 5921. DOI: 10.1038/s41467-020-19097-x doi: 10.1038/s41467-020-19097-x

[59]	FOZOUNI P, SON S, DE LEÓN DERBY M D, et al. Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy[J]. Cell, 2021, 184(2): 323-333. e9. DOI: 10.1016/j.cell.2020.12.001 doi: 10.1016/j.cell.2020.12.001

[60]	CHANG Y F, DENG Y, LI T Y, et al. Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a[J]. Transboundary and Emerging Diseases, 2020, 67(2): 564-571. DOI: 10.1111/tbed.13368 doi: 10.1111/tbed.13368

[61]	TIAN T, QIU Z Q, JIANG Y Z, et al. Exploiting the orthogonal CRISPR-Cas12a/Cas13a trans-cleavage for dual-gene virus detection using a handheld device[J]. Biosensors & Bioelectronics, 2022, 196: 113701. DOI: 10.1016/j.bios.2021.113701 doi: 10.1016/j.bios.2021.113701

[62]	HUANG M Q, ZHOU X M, WANG H Y, et al. Clustered regularly interspaced short palindromic repeats/Cas9 triggered isothermal amplification for site-specific nucleic acid detection[J]. Analytical Chemistry, 2018, 90(3): 2193-2200. DOI: 10.1021/acs.analchem.7b04542 doi: 10.1021/acs.analchem.7b04542

[63]	WANG L Y, SHEN X Y, WANG T, et al. A lateral flow strip combined with Cas9 nickase-triggered amplification reaction for dual food-borne pathogen detection[J]. Biosensors & Bioelectronics, 2020, 165: 112364. DOI: 10.1016/j.bios.2020.112364 doi: 10.1016/j.bios.2020.112364

[64]	WANG X S, XIONG E H, TIAN T, et al. Clustered regularly interspaced short palindromic repeats/Cas9-mediated lateral flow nucleic acid assay[J]. ACS Nano, 2020, 14(2): 2497-2508. DOI: 10.1021/acsnano.0c00022 doi: 10.1021/acsnano.0c00022

[65]	GUK K, KEEM J O, HWANG S G, et al. A facile, rapid and sensitive detection of MRSA using a CRISPR-mediated DNA FISH method, antibody-like dCas9/sgRNA complex[J]. Biosensors & Bioelectronics, 2017, 95: 67-71. DOI: 10.1016/j.bios.2017.04.016 doi: 10.1016/j.bios.2017.04.016

[66]	PENG L, ZHOU J, YIN L J, et al. Integration of logic gates to CRISPR/Cas12a system for rapid and sensitive detection of pathogenic bacterial genes[J]. Analytica Chimica Acta, 2020, 1125: 162-168. DOI: 10.1016/j.aca.2020.05.017 doi: 10.1016/j.aca.2020.05.017

[67]	BAI J, LIN H S, LI H J, et al. Cas12a-based on-site and rapid nucleic acid detection of African swine fever[J]. Frontiers in Microbiology, 2019, 10: 2830. DOI: 10.3389/fmicb.2019.02830 doi: 10.3389/fmicb.2019.02830

[68]	ZHANG W Y, XU L, LIU Q, et al. Enzymatic recombinase amplification coupled with CRISPR-Cas12a for ultrasensitive, rapid, and specific Porcine circovirus 3 detection[J]. Molecular and Cellular Probes, 2021, 59: 101763. DOI: 10.1016/j.mcp.2021.101763 doi: 10.1016/j.mcp.2021.101763

[69]	SUKONTA T, SENAPIN S, MEEMETTA W, et al. CRISPR-based platform for rapid, sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)[J]. Journal of Fish Diseases, 2022, 45(1): 107-120. DOI: 10.1111/jfd.13541 doi: 10.1111/jfd.13541

[70]	WU H, CHEN Y J, YANG Q Q, et al. A reversible valve-assisted chip coupling with integrated sample treatment and CRISPR/Cas12a for visual detection of Vibrio parahaemoly-ticus [J]. Biosensors & Bioelectronics, 2021, 188: 113352. DOI: 10.1016/j.bios.2021.113352 doi: 10.1016/j.bios.2021.113352

[71]	YUAN C Q, TIAN T, SUN J, et al. Universal and naked-eye gene detection platform based on the clustered regularly interspaced short palindromic repeats/Cas12a/13a system[J]. Analytical Chemistry, 2020, 92(5): 4029-4037. DOI: 10.1021/acs.analchem.9b05597 doi: 10.1021/acs.analchem.9b05597

[72]	KHAN H, KHAN A, LIU Y F, et al. CRISPR-Cas13a mediated nanosystem for attomolar detection of canine parvovirus type 2[J]. Chinese Chemical Letters, 2019, 30(12): 2201-2204. DOI: 10.1016/j.cclet.2019.10.032 doi: 10.1016/j.cclet.2019.10.032

[73]	SHEN J J, ZHOU X M, SHAN Y Y, et al. Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction[J]. Nature Communications, 2020, 11: 267. DOI: 10.1038/s41467-019-14135-9 doi: 10.1038/s41467-019-14135-9

[74]	ZHOU J, YIN L J, DONG Y N, et al. CRISPR-Cas13a based bacterial detection platform: sensing pathogen Staphylococcus aureus in food samples[J]. Analytica Chimica Acta, 2020, 1127: 225-233. DOI: 10.1016/j.aca.2020.06.041 doi: 10.1016/j.aca.2020.06.041

[75]	ZHANG T, ZHOU W H, LIN X Y, et al. Light-up RNA aptamer signaling-CRISPR-Cas13a-based mix-and-read assays for profiling viable pathogenic bacteria[J]. Biosensors & Bioelectronics, 2021, 176: 112906. DOI: 10.1016/j.bios.2020.112906 李艳，浙江大学动物科学学院“百人计划”研究员，博士生导师。中国科学院武汉病毒研究所和美国纽约州卫生部Wadsworth中心联合培养博士，2010年获理学博士学位。2011年起师从美国科学院院士Nancy Speck教授进行博士后研究。2017年10月加盟浙江大学动物科学学院动物医学系，研究方向为兽医病理与比较生物医学。近年来在SCI收录期刊共发表论文30余篇，其中在Blood、Genes & Development等期刊以第一作者或者通信作者发表论文17篇。作为主要完成人之一获得2018年国家自然科学奖二等奖。主持国家重点研发计划政府间国际科技创新合作项目、国家自然科学基金面上项目和青年项目、浙江省自然科学基金重点项目、海南省自然科学基金面上项目和海南省科技计划三亚崖州湾科技城联合项目。 doi: 10.1016/j.bios.2020.112906

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