Ⅱ类CRISPR/Cas系统及其在细菌合成生物学中的应用

doi:10.11843/j.issn.0366-6964.2025.04.012

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (4): 1608-1620.doi: 10.11843/j.issn.0366-6964.2025.04.012

Ⅱ类CRISPR/Cas系统及其在细菌合成生物学中的应用

李晓晗¹^,²(), 李桂萍²(), 霍彩云², 张启龙³, 孙英健¹, 孙惠玲²^,*()

1. 北京农学院动物科学技术学院, 北京 100096
2. 北京市农林科学院畜牧兽医研究所, 北京 100097
3. 北京市动物疫病预防控制中心, 北京 102629

收稿日期:2024-05-27 出版日期:2025-04-23 发布日期:2025-04-28
通讯作者: 孙惠玲 E-mail:1025290934@qq.com;lgp709@sina.cn;sunhuiling01@163.com
作者简介:李晓晗(1998-), 女, 河北承德人, 硕士生, 主要从事动物病原微生物与免疫研究, E-mail: 1025290934@qq.com
李桂萍(1973-), 女, 北京人, 大学, 主要从事动物传染病防控研究, E-mail: lgp709@sina.cn
第一联系人：
李晓晗和李桂萍为同等贡献作者
基金资助:
北京市农林科学院改革和发展(XMS202409)

Class II CRISPR/Cas Systems and Their Applications in Bacterial Synthetic Biology

LI Xiaohan¹^,²(), LI Guiping²(), HUO Caiyun², ZHANG Qilong³, SUN Yingjian¹, SUN Huiling²^,*()

1. College of Animal Science and Technology, Beijing University of Agriculture, Beijing 100096, China
2. Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
3. Beijing Center for Animal Disease Control and Prevention, Beijing 102629, China

Received:2024-05-27 Online:2025-04-23 Published:2025-04-28
Contact: SUN Huiling E-mail:1025290934@qq.com;lgp709@sina.cn;sunhuiling01@163.com

摘要/Abstract

摘要：

CRISPR/Cas系统是一种广泛存在于细菌和古细菌中的适应性免疫系统，通过RNA介导的核酸内切酶靶向识别并切割特定的核酸片段，以抵御外来核酸入侵。根据效应蛋白复合物的组成不同，CRISPR/Cas系统可分为两大类：Ⅰ类CRISPR/Cas系统利用多个Cas蛋白组成的效应复合物与crRNA共同作用来发挥靶链切割功能，而Ⅱ类CRISPR/Cas系统则由单个多结构域蛋白组成效应子模块。其中，单一组分效应蛋白介导的Ⅱ类CRISPR基因编辑技术相对简便，近年来已被广泛应用于细菌基因表达调控、遗传修饰、代谢途径优化以及病原微生物检测等合成生物学相关领域。根据Cas效应蛋白核酸酶结构域的差异，Ⅱ类系统又可进一步分为Ⅱ型、V型和VI型三个亚型，不同亚型的系统在细菌合成生物学中的应用也存在差异。本文综述了Ⅱ类CRISPR-Cas系统的免疫学机制、分类与特点，及其在工业细菌中的应用现状与最新进展，旨在为未来细菌合成生物学研究中选择、优化和探索更多的CRISPR/Cas系统提供参考。

关键词: CRISPR/Cas系统, 细菌, 基因编辑, 合成生物学, 效应蛋白

Abstract:

The CRISPR/Cas system is an adaptive immune system widely present in bacteria and archaea. It targets and cleaves specific nucleic acid sequences using RNA-guided nucleases to defend against foreign nucleic acid invasion. Based on the composition of effector protein complexes, the CRISPR/Cas system can be divided into two main classes: Class I systems utilize effector complexes composed of multiple Cas proteins, acting together with crRNA to exert target cleavage function, while Class II systems consist of single, multi-domain protein effector modules. Among them, the single-component effector protein-mediated Class II CRISPR gene editing technology is relatively straightforward and has been widely applied in synthetic biology fields such as bacterial gene expression regulation, genetic modification, metabolic pathway optimization, and pathogenic microorganism detection. Based on the differences in the Cas effector protein nuclease domains, Class II systems can further be categorized into subtypes II, V, and VI, each exhibiting differences in their applications in bacterial synthetic biology. This article reviews the immunological mechanisms, classification, and characteristics of Class II CRISPR-Cas systems, as well as their current applications and the latest developments in industrial bacteria, aiming to provide a reference for future research in bacterial synthetic biology to select, optimize, and explore more CRISPR/Cas systems.

Key words: CRISPR/Cas system, bacteria, gene editing, synthetic biology, effector protein

中图分类号:

李晓晗, 李桂萍, 霍彩云, 张启龙, 孙英健, 孙惠玲. Ⅱ类CRISPR/Cas系统及其在细菌合成生物学中的应用[J]. 畜牧兽医学报, 2025, 56(4): 1608-1620.

LI Xiaohan, LI Guiping, HUO Caiyun, ZHANG Qilong, SUN Yingjian, SUN Huiling. Class II CRISPR/Cas Systems and Their Applications in Bacterial Synthetic Biology[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(4): 1608-1620.

图/表 6

表 1

图 1

图 2

图 3

图 4

图 5

参考文献 69

1	CHANDRASEGARAN S , CARROLL D . Origins of programmable nucleases for genome engineering[J]. J Mol Biol, 2016, 428 (5): 963- 989. doi: 10.1016/j.jmb.2015.10.014
2	DEVEAU H , GARNEAU J E , MOINEAU S . CRISPR/Cas system and its role in phage-bacteria interactions[J]. Annu Rev Microbiol, 2010, 64, 475- 493. doi: 10.1146/annurev.micro.112408.134123
3	ISHINO Y , SHINAGAWA H , MAKINO K , et al. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product[J]. J Bacteriol, 1987, 169 (12): 5429- 5433. doi: 10.1128/jb.169.12.5429-5433.1987
4	JANSEN R , VAN EMBDEN J D A , GAASTRA W , et al. Identification of genes that are associated with DNA repeats in prokaryotes[J]. Mol Microbiol, 2002, 43 (6): 1565- 1575. doi: 10.1046/j.1365-2958.2002.02839.x
5	JINEK M , CHYLINSKI K , FONFARA I , et al. A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337 (6096): 816- 821. doi: 10.1126/science.1225829
6	MAKAROVA K S , HAFT D H , BARRANGOU R , et al. Evolution and classification of the CRISPR-Cas systems[J]. Nat Rev Microbiol, 2011, 9 (6): 467- 477. doi: 10.1038/nrmicro2577
7	MAKAROVA K S , WOLF Y I , KOONIN E V . The basic building blocks and evolution of CRISPR-Cas systems[J]. Biochem Soc Trans, 2013, 41 (6): 1392- 1400. doi: 10.1042/BST20130038
8	MAKAROVA K S , WOLF Y I , ALKHNBASHI O S , et al. An updated evolutionary classification of CRISPR-Cas systems[J]. Nat Rev Microbiol, 2015, 13 (11): 722- 736. doi: 10.1038/nrmicro3569
9	ZHAO J , FANG H , ZHANG D W . Expanding application of CRISPR-Cas9 system in microorganisms[J]. Synth Syst Biotechnol, 2020, 5 (4): 269- 276. doi: 10.1016/j.synbio.2020.08.001
10	TAKEDA S N , NAKAGAWA R , OKAZAKI S , et al. Structure of the miniature type V-F CRISPR-Cas effector enzyme[J]. Mol Cell, 2021, 81 (3): 558- 570. doi: 10.1016/j.molcel.2020.11.035
11	GAO P , YANG H , RAJASHANKAR K R , et al. Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition[J]. Cell Res, 2016, 26 (8): 901- 913. doi: 10.1038/cr.2016.88
12	VAN BELJOUW S P B , SANDERS J , RODRÍGUEZ-MOLINA A , et al. RNA-targeting CRISPR-Cas systems[J]. Nat Rev Microbiol, 2023, 21 (1): 21- 34. doi: 10.1038/s41579-022-00793-y
13	SHMAKOV S , ABUDAYYEH O O , MAKAROVA K S , et al. Discovery and functional characterization of diverse class 2 CRISPR-Cas systems[J]. Mol Cell, 2015, 60 (3): 385- 397. doi: 10.1016/j.molcel.2015.10.008
14	GARNEAU J E , DUPUIS M È , VILLION M , et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA[J]. Nature, 2010, 468 (7320): 67- 71. doi: 10.1038/nature09523
15	CHYLINSKI K , MAKAROVA K S , CHARPENTIER E , et al. Classification and evolution of type II CRISPR-Cas systems[J]. Nucleic Acids Res, 2014, 42 (10): 6091- 6105. doi: 10.1093/nar/gku241
16	LIEBER M R . The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway[J]. Annu Rev Biochem, 2010, 79, 181- 211. doi: 10.1146/annurev.biochem.052308.093131
17	ZHANG J P , LI X L , LI G H , et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage[J]. Genome Biol, 2017, 18 (1): 35. doi: 10.1186/s13059-017-1164-8
18	HIDALGO-CANTABRANA C , GOH Y J , BARRANGOU R . Characterization and repurposing of type I and type II CRISPR-Cas systems in bacteria[J]. J Mol Biol, 2019, 431 (1): 21- 33. doi: 10.1016/j.jmb.2018.09.013
19	HELER R , SAMAI P , MODELL J W , et al. Cas9 specifies functional viral targets during CRISPR-Cas adaptation[J]. Nature, 2015, 519 (7542): 199- 202. doi: 10.1038/nature14245
20	LARSON M H , GILBERT L A , WANG X W , et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression[J]. Nat Protoc, 2013, 8 (11): 2180- 2196. doi: 10.1038/nprot.2013.132
21	CLETO S , JENSEN J V K , WENDISCH V F , et al. Corynebacterium glutamicum metabolic engineering with CRISPR interference (CRISPRi)[J]. ACS Synth Biol, 2016, 5 (5): 375- 385. doi: 10.1021/acssynbio.5b00216
22	SONG X , HUANG H , XIONG Z Q , et al. CRISPR-Cas9^D10A nickase-assisted genome editing in Lactobacillus casei[J]. Appl Environ Microbiol, 2017, 83 (22): e01259- 17.
23	LIU Z Q , DONG H N , CUI Y L , et al. Application of different types of CRISPR/Cas-based systems in bacteria[J]. Microb Cell Fact, 2020, 19 (1): 172. doi: 10.1186/s12934-020-01431-z
24	LIU Y , WAN X Y , WANG B J . Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria[J]. Nat Commun, 2019, 10 (1): 3693. doi: 10.1038/s41467-019-11479-0
25	GOH Y J , BARRANGOU R . Portable CRISPR-Cas9^N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei[J]. Appl Environ Microbiol, 2021, 87 (6): e02669- 20.
26	CHEN Q L , WANG B , PAN L . Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis via CRISPR/Cas9n and its immobilization[J]. Appl Microbiol Biotechnol, 2024, 108 (1): 149. doi: 10.1007/s00253-023-12889-3
27	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
28	ZHONG Z H , ZHANG Y X , YOU Q , et al. Plant genome editing using FnCpf1 and LbCpf1 Nucleases at redefined and altered PAM sites[J]. Mol Plant, 2018, 11 (7): 999- 1002. doi: 10.1016/j.molp.2018.03.008
29	SAFARI F , ZARE K , NEGAHDARIPOUR M , et al. CRISPR Cpf1 proteins: structure, function and implications for genome editing[J]. Cell Biosci, 2019, 9, 36. doi: 10.1186/s13578-019-0298-7
30	ZHU X W , WU Y K , LV K Q , et al. Combining CRISPR-Cpf1 and recombineering facilitates fast and efficient genome editing in Escherichia coli[J]. ACS Synth Biol, 2022, 11 (5): 1897- 1907. doi: 10.1021/acssynbio.2c00041
31	ZHANG Y , YUAN J F . CRISPR/Cas12a-mediated genome engineering in the photosynthetic bacterium Rhodobacter capsulatus[J]. Microb Biotechnol, 2021, 14 (6): 2700- 2710. doi: 10.1111/1751-7915.13805
32	KNOOT C J , BISWAS S , PAKRASI H B . Tunable repression of key photosynthetic processes using Cas12a CRISPR interference in the fast-growing cyanobacterium Synechococcus sp. UTEX 2973[J]. ACS Synth Biol, 2020, 9 (1): 132- 143. doi: 10.1021/acssynbio.9b00417
33	YOU Y , ZHANG P P , WU G S , et al. Highly specific and sensitive detection of Yersinia pestis by portable Cas12a-UPTLFA platform[J]. Front Microbiol, 2021, 12, 700016. doi: 10.3389/fmicb.2021.700016
34	SAM I K , CHEN Y Y , MA J , et al. TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis[J]. J Infect, 2021, 83 (1): 54- 60. doi: 10.1016/j.jinf.2021.04.032
35	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
36	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
37	GARNER K L . Principles of synthetic biology[J]. Essays Biochem, 2021, 65 (5): 791- 811. doi: 10.1042/EBC20200059
38	蒋成辉, 曾巧英, 王萌, 等. CRISPR/Cas9构建srtA基因敲除的金黄色葡萄球菌[J]. 生物技术通报, 2020, 36 (9): 253- 265.
	JIANG C H , ZENG Q Y , WANG M , et al. Construction of srtA-knockout strain in Staphylococcus aureus by CRISPR/Cas9 technology[J]. Biotechnology Bulletin, 2020, 36 (9): 253- 265.
39	杨茗崴, 王玮玉, 王裕光, 等. fimH基因敲除对大肠杆菌NMGCF-19菌株致病性的影响[J]. 中国兽医学报, 2023, 43 (9): 1881- 1887.
	YANG M W , WANG W Y , WANG Y G , et al. Effect of fimH gene knockout on the pathogenicity of Escherichia coli NMGCF-19 strain[J]. Chinese Journal of Veterinary Science, 2023, 43 (9): 1881- 1887.
40	何京桦, 乐科易. 利用CRISPR/Cas9系统敲除大肠杆菌tnaA基因[J]. 生物学杂志, 2019, 36 (6): 17- 19.
	HE J H , LE K Y . CRISPR/ Cas9-mediated tnaA knockout in Escherichia coli[J]. Journal of Biology, 2019, 36 (6): 17- 19.
41	RAINHA J , RODRIGUES J L , RODRIGUES L R . CRISPR-Cas9:a powerful tool to efficiently engineer Saccharomyces cerevisiae[J]. Life (Basel), 2020, 11 (1): 13.
42	GÖTTL V L , SCHMITT I , BRAUN K , et al. CRISPRi-library-guided target identification for engineering carotenoid production by Corynebacterium glutamicum[J]. Microorganisms, 2021, 9 (4): 670. doi: 10.3390/microorganisms9040670
43	XIN Q L , CHEN Y D , CHEN Q L , et al. Development and application of a fast and efficient CRISPR-based genetic toolkit in Bacillus amyloliquefaciens LB1ba02[J]. Microb Cell Fact, 2022, 21 (1): 99. doi: 10.1186/s12934-022-01832-2
44	AMERUOSO A , VILLEGAS KCAM M C , COHEN K P , et al. Activating natural product synthesis using CRISPR interference and activation systems in Streptomyces[J]. Nucleic Acids Res, 2022, 50 (13): 7751- 7760. doi: 10.1093/nar/gkac556
45	张博, 杨玉凤, 贺周霖, 等. 利用CRISPRi挖掘大肠杆菌生物合成D-泛酸的关键基因[J]. 微生物学报, 2023, 63 (12): 4625- 4643.
	ZHANG B , YANG Y F , HE Z L , et al. Mining key genes for biosynthesis of D-pantothenic acid in Escherichia coli by CRISPRi[J]. Acta Microbiologica Sinica, 2023, 63 (12): 4625- 4643.
46	HAO M J , HE Y Z , ZHANG H F , et al. CRISPR-Cas9-mediated carbapenemase gene and plasmid curing in carbapenem-resistant Enterobacteriaceae[J]. Antimicrob Agents Chemother, 2020, 64 (9): e00843- 20.
47	WU X , ZHA J , KOFFAS M A G , et al. Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR-dCas9[J]. Biotechnol Bioeng, 2019, 116 (12): 3149- 3159. doi: 10.1002/bit.27143
48	WAN X L , LI Q Y , OLSEN R H , et al. Engineering a CRISPR interference system targeting AcrAB-TolC efflux pump to prevent multidrug resistance development in Escherichia coli[J]. J Antimicrob Chemother, 2022, 77 (8): 2158- 2166. doi: 10.1093/jac/dkac166
49	CHEN S Z , CAO H L , XU Z R , et al. A type I-F CRISPRi system unveils the novel role of CzcR in modulating multidrug resistance of Pseudomonas aeruginosa[J]. Microbiol Spectr, 2023, 11 (5): e01123- 23.
50	GU T N , ZHAO S Q , PI Y S , et al. Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase[J]. Chem Sci, 2018, 9 (12): 3248- 3253. doi: 10.1039/C8SC00637G
51	吕言, 任晓昕, 徐大庆. CRISPR/Cas9和λ-Red基因敲除技术在大肠杆菌中的应用比较[J]. 微生物前沿, 2022, 11 (1): 1- 10.
	LÜ Y , REN X X , XU D Q . Comparative study of CRISPR/Cas9 and λ-red gene knockout techniques in Eschrichia coli[J]. Advances in Microbiology, 2022, 11 (1): 1- 10.
52	JIANG Y , CHEN B , DUAN C L , et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system[J]. Appl Environ Microbiol, 2015, 81 (7): 2506- 2514. doi: 10.1128/AEM.04023-14
53	SU B L , SONG D D , ZHU H H . Homology-dependent recombination of large synthetic pathways into E. coli genome via λ-Red and CRISPR/Cas9 dependent selection methodology[J]. Microb Cell Fact, 2020, 19 (1): 108. doi: 10.1186/s12934-020-01360-x
54	ZHAO R , LIU Y Q , ZHANG H , et al. CRISPR-Cas12a-mediated gene deletion and regulation in Clostridium ljungdahlii and its application in carbon flux redirection in synthesis gas fermentation[J]. ACS Synth Biol, 2019, 8 (10): 2270- 2279. doi: 10.1021/acssynbio.9b00033
55	KO Y J , CHA J , JEONG W Y , et al. Bio-isopropanol production in Corynebacterium glutamicum: metabolic redesign of synthetic bypasses and two-stage fermentation with gas stripping[J]. Bioresour Technol, 2022, 354, 127171. doi: 10.1016/j.biortech.2022.127171
56	YAN M Y , YAN H Q , REN G X , et al. CRISPR-Cas12a-assisted recombineering in bacteria[J]. Appl Environ Microbiol, 2017, 83 (17): e00947- 17.
57	AO X , YAO Y , LI T , et al. A multiplex genome editing method for Escherichia coli based on CRISPR-Cas12a[J]. Front Microbiol, 2018, 9, 2307. doi: 10.3389/fmicb.2018.02307
58	黄紫蕾, 龙腾飞, 刘雅红, 等. 基于CRISPR-Cas12f对抗耐药革兰氏阴性菌的新型递送系统[C]//中国畜牧兽医学会兽医药理毒理学分会第十三次全国会员代表大会暨第十七次学术研讨会论文集. 南宁: 中国畜牧兽医学会兽医药理毒理学分会, 2023: 1.
	HUANG Z L, LONG T F, LIU Y H, et al. A novel delivery system based on CRISPR-Cas12f for combating antibiotic-resistant Gram-negative bacteria[C]//Proceedings of the Chinese Association of Animal Science and Veterinary Medicine. Nanning: Chinese Society of Animal Husbandry and Veterinary Medicine Veterinary Pharmacology and Toxicology Branch, 2023: 1. (in Chinese)
59	AI J W , ZHOU X , XU T , et al. CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis[J]. Emerg Microbes Infect, 2019, 8 (1): 1361- 1369. doi: 10.1080/22221751.2019.1664939
60	LI Y , DENG F , HALL T , et al. CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader[J]. Water Res, 2021, 203, 117553. doi: 10.1016/j.watres.2021.117553
61	冀霞, 代绍密, 余若菁, 等. CRISPR-Cas12a检测牛奶中大肠杆菌方法的建立与评价[J]. 食品研究与开发, 2023, 44 (18): 179- 184. doi: 10.12161/j.issn.1005-6521.2023.18.024
	JI X , DAI S M , YU R J , et al. Establishment and evaluation of CRISPR-Cas12a method for detecting Escherichia coli in milk[J]. Food Research and Development, 2023, 44 (18): 179- 184. doi: 10.12161/j.issn.1005-6521.2023.18.024
62	HAO J , XIE L F , YANG T M , et al. Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification[J]. Talanta, 2023, 255, 124220. doi: 10.1016/j.talanta.2022.124220
63	LU L Y , ZHANG H M , LIN F H , et al. Sensitive and automated detection of bacteria by CRISPR/Cas12a-assisted amplification with digital microfluidics[J]. Sens Actuators B: Chem, 2023, 381, 133409. doi: 10.1016/j.snb.2023.133409
64	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
65	KIGA K , TAN X E , IBARRA-CHÁVEZ R , et al. Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria[J]. Nat Commun, 2020, 11 (1): 2934. doi: 10.1038/s41467-020-16731-6
66	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
67	ZHOU J , YIN L J , DONG Y N , et al. CRISPR-Cas13a based bacterial detection platform: sensing pathogen Staphylococcus aureus in food samples[J]. Anal Chim Acta, 2020, 1127, 225- 233. doi: 10.1016/j.aca.2020.06.041
68	LIU J , CARMICHAEL C , HASTURK H , et al. Rapid specific detection of oral bacteria using Cas13-based SHERLOCK[J]. J Oral Microbiol, 2023, 15 (1): 2207336. doi: 10.1080/20002297.2023.2207336
69	侯雅超, 邢微微, 王亚楠, 等. 针对副溶血性弧菌toxR基因的RPA-CRISPR/Cas13a荧光快速检测方法的建立及应用[J]. 中华医院感染学杂志, 2024, 34 (14): 2081- 2086.
	HOU Y C , XING W W , WANG Y N , et al. Establishment and application of RPA-CRISPR/Cas13a fluorescent rapid assay targeting Vibrio parahaemolyticus toxR gene[J]. Chinese Journal of Nosocomiology, 2024, 34 (14): 2081- 2086.

类型 Type	Ⅱ型 Type Ⅱ	Ⅴ型 Type Ⅴ	Ⅵ型 Type Ⅵ
标志性效应蛋白 Signature effector protein	Cas9，II-A	Cas12a，V-A	Cas13a，VI-A
发源菌 Bacteria of origin	化脓性链球菌	新凶手弗朗西斯菌氨基酸球菌	纤毛菌瘤胃球菌
		氨基酸球菌	瘤胃球菌
		毛螺菌	普雷沃菌
		摩拉菌	李斯特菌
CRISPR-RNA	sgRNA，100 nt	crRNA，42~44 nt	crRNA，52~66 nt
PAM序列	G富集	T富集	无
PAM sequence	5′-NGG-3′	5′-TT(T)N-3′	PFS序列
核酸酶结构域Nuclease domain	HNH、RuvC	RuvC	HEPN
切割方式Cutting mode	dsDNA，平末端	dsDNA，黏末端	ssRNA
RNA酶活性RNAse activity	无	有	有

[1]	解雅茹, 金昊延, 孔辰, 蔡蓓, 张令锴. CRISPR/Cas9系统在家畜生殖细胞中的研究进展[J]. 畜牧兽医学报, 2025, 56(2): 479-491.
[2]	包斌武, 邹惠影, 李俊良, 高晨, 高会江, 杜振伟, 张博玉, 李俊雅, 高雪. 基因编辑技术的研究进展[J]. 畜牧兽医学报, 2025, 56(1): 1-14.
[3]	张蕾, 陈亮, 冯万宇, 兰世捷, 苗艳, 田秋丰, 白长胜, 张备, 董佳强, 江波涛, 王洪宝, 史同瑞, 黄宣凯. 生物被膜在动物细菌感染致病机理中的作用[J]. 畜牧兽医学报, 2025, 56(1): 107-114.
[4]	刘雯雯, 董发明, 毕延震. 多基因编辑技术的发展及其在畜牧种质创新中的应用[J]. 畜牧兽医学报, 2024, 55(8): 3267-3275.
[5]	梁瑞英, 索静霞, 梁琳, 刘贤勇, 丁家波, 索勋, 汤新明. 艾美耳球虫的遗传操作：平台建立、应用与展望[J]. 畜牧兽医学报, 2024, 55(8): 3362-3373.
[6]	黄杰, 阮子豪, 蔡瑞. 抗菌肽在猪精液常温保存中的应用研究进展[J]. 畜牧兽医学报, 2024, 55(4): 1401-1411.
[7]	高远集, 刘畅, 陈淼, 陈松彪, 张俊峰, 李静, 贾艳艳, 廖成水, 郭荣显, 丁轲, 余祖华, 尚珂. 细菌外膜囊泡结构、分泌特性及致病机制[J]. 畜牧兽医学报, 2024, 55(3): 971-983.
[8]	张多, 滕蔓, 张卓, 刘金玲, 郑鹿平, 各思雨, 韩放, 罗琴, 柴书军, 赵东, 余祖华, 罗俊. 一株马立克病病毒特超强变异株meq基因编辑缺失候选疫苗毒株的构建与鉴定[J]. 畜牧兽医学报, 2024, 55(12): 5672-5683.
[9]	张学富, 陈运通, 范文瑞, 张子博, 于蒙蒙, 王素艳, 祁小乐, 李留安, 高玉龙. 鸡chNHE1精准基因编辑细胞系的构建及其抗ALV-J感染的研究[J]. 畜牧兽医学报, 2024, 55(11): 5238-5246.
[10]	安一娜, 谭姝瑜, 冯岚迪, 鲍明悦, 刘梦晗, 尹勤昊, 董彦君. 鸢尾素对感染大肠埃希菌小鼠组织损伤及细菌清除的影响[J]. 畜牧兽医学报, 2024, 55(11): 5267-5277.
[11]	丁修虎, 林志平, 赵芳, 陈坤琳, 仲跻峰, 张燕, 高运东, 李惠侠, 王慧利, 张建丽, 丁强. 利用CRISPR/Cas9技术制备BLG基因敲除牛乳腺上皮细胞系[J]. 畜牧兽医学报, 2024, 55(10): 4475-4488.
[12]	付涵, 卢冲, 缪荣浩, 卢亚宾, 李建龙, 刘建华, 耿明阳, 郭庆勇, 买占海, 况玲. 流产对母马阴道和肠道菌群多样性的影响及阴道细菌的分离鉴定[J]. 畜牧兽医学报, 2024, 55(10): 4700-4719.
[13]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[14]	于秀菊, 张敏爱, 胡燕姣, 朱芷葳, 王海东, 杨丽华, 范阔海. 枯草芽孢杆菌细菌素的分离、表达及稳定性分析[J]. 畜牧兽医学报, 2024, 55(1): 323-333.
[15]	米慧, 彭灿, 贺志雄, 谭支良. 基于流式细胞术分选绵羊分泌型IgA包裹的消化道细菌[J]. 畜牧兽医学报, 2023, 54(7): 2924-2931.

Ⅱ类CRISPR/Cas系统及其在细菌合成生物学中的应用

Class II CRISPR/Cas Systems and Their Applications in Bacterial Synthetic Biology

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 69

相关文章 15

编辑推荐

Metrics

本文评价