CRISPR­Cas系统在枯草芽孢杆菌基因组编辑中的研究进展

doi:10.13304/j.nykjdb.2023.0260

摘要/Abstract

摘要：

枯草芽孢杆菌（Bacillus subtilis）是一种食品安全级微生物，现已广泛应用于工业发酵。CRISPR（clustered regularly interspaced short palindromic repeats）介导的基因组编辑技术在以枯草芽孢杆菌为底盘细胞的微生物代谢工程研究中发挥了重要作用。介绍了CRISPR-Cas系统的免疫应答机制和分类以及枯草芽孢杆菌中CRISPR-Cas9基因组编辑的3种类型，重点总结了最新的CRISPR开发和设计策略，以期为优化现有的枯草芽孢杆菌基因组编辑系统提供参考，从而提高枯草芽孢杆菌的工业化应用潜能。

关键词: 枯草芽孢杆菌, 基因编辑, CRISPR-Cas9, CRISPR-Cpf1, PAM无依赖

Abstract:

Bacillus subtilis is a food safety microorganism， which has been widely used in industrial fermentation. CRISPR （clustered regularly interspaced short palindromic repeats） mediated genome editing technology has played an important role in the research of microbial metabolic engineering with B. subtilis as the chassis cell. The immune response mechanism and classification of CRISPR-Cas system were introduced， as well as the 3 types of CRISPR-Cas9 genome editing in B. subtilis. It focused on summarizing the latest CRISPR development and design strategies， with a view to providing references for optimizing existing B. subtilis genome editing systems， thereby improving the industrial application potential of B. subtilis.

Key words: Bacillus subtilis, genome editing, CRISPR-Cas9, CRISPR-Cpf1, PAM-independent

中图分类号:

Q812

孙志康, 李力群, 郝捷, 吴晗, 吴娜, 郑超, 季嫱, 李选文, 陈晨. CRISPRCas系统在枯草芽孢杆菌基因组编辑中的研究进展[J]. 中国农业科技导报, 2025, 27(2): 24-32.

Zhikang SUN, Liqun LI, Jie HAO, Han WU, Na WU, Chao ZHENG, Qiang JI, Xuanwen LI, Chen CHEN. Recent Advances of CRISPR-Cas System in Genome Editing of Bacillus subtilis[J]. Journal of Agricultural Science and Technology, 2025, 27(2): 24-32.

图/表 2

参考文献 58

1	EARL A M, LOSICK R, KOLTER R. Ecology and genomics of Bacillus subtilis [J]. Trends Microbiol., 2008, 16(6):269-275.
2	DEUTSCHER J. The mechanisms of carbon catabolite repression in bacteria [J]. Curr. Opin. Microbiol., 2008, 11(2):87-93.
3	NICOLAS P, MÄDER U, DERVYN E, et al.. Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis [J]. Science, 2012, 335(6072):1103-1106.
4	CHEN J Q, ZHAO L Q, FU G, et al.. A novel strategy for protein production using non-classical secretion pathway in Bacillus subtilis [J/OL]. Microb. Cell Fact., 2016, 15(1):1-16 [2023-03-03]. .
5	LIU Y, LIU L, LI J, et al.. Synthetic biology toolbox and chassis development in Bacillus subtilis [J]. Trends Biotechnol., 2019, 37(5):548-562.
6	GU Y, XU X H, WU Y K, et al.. Advances and prospects of Bacillus subtilis factories: from rational design to industrial applications [J]. Metab. Eng., 2018, 50(5):109-121.
7	XIANG M J, KANG Q, ZHANG D W. Advances on systems metabolic engineering of Bacillus subtilis as a chassis cell [J]. Syn. Syst. Biotechnol., 2020, 5(4):245-251.
8	WIDNER B, BEHR R, VON DOLLEN S, et al.. Hyaluronic acid production in Bacillus subtilis [J]. Appl. Environ. Microbiol., 2005, 71(7):3747-3752.
9	CUI S X, LV X Q, WU Y K, et al.. Engineering a bifunctional Phr60-Rap60-Spo0A quorum-sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis [J]. ACS Synth. Biol., 2019, 8(8):1826-1837.
10	LIU Y F, LIU L, SHIN H D, et al.. Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine [J]. Metab. Eng., 2013, 19:107-115.
11	张续,班睿,刘露,等.枯草芽孢杆菌基因修饰生产核黄素[J].微生物学通报,2017,44(1):59-67.
	ZHANG X, BAN R, LIU L, et al.. Riboflavin production by a genetically modified Bacillus subtilis [J]. Microbiol. China, 2017, 44(1):59-67.
12	DENG J Y, CHEN C M, GU Y, et al.. Creating an in vivo bifunctional gene expression circuit through an aptamer-based regulatory mechanism for dynamic metabolic engineering in Bacillus subtilis [J]. Metab. Eng., 2019, 55:179-190.
13	DEB S, CHOUDHURY A, KHARBYNGAR B, et al.. Applications of CRISPR/Cas9 technology for modification of the plant genome [J]. Genetica, 2022, 150(1):1-12.
14	林璐,吕雪琴,林延峰,等.枯草芽孢杆菌底盘细胞的设计、构建及应用[J].合成生物学,2020,1(2):247-265.
	LIN L, LYU X Q, LIN Y F, et al.. Advances in design, construction and applications of Bacillus subtilis chassis cells [J]. Synth. Biol. J., 2020, 1(2):247-265.
15	ZHANG K, DUAN X, WU J. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system [J/OL]. Sci. Rep., 2016, 6(1):27943 [2023-03-03]. .
16	张春晓,王丽丽,陈赟,等.β-甘露聚糖酶突变体食品级枯草芽孢杆菌表达载体、表达系统、构建方法和应用:CN114703163A [P].2022-07-05.
17	MAKAROVA K S, WOLF Y I, IRANZO J, et al.. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants [J]. Nat. Rev. Microbiol., 2020, 18(2):67-83.
18	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.
19	LI T X, YANG Y Y, QI H Z, et al.. CRISPR/Cas9 therapeutics: progress and prospects [J/OL]. Signal. Transduct. Tar., 2023, 8(1):36 [2023-04-23]. .
20	吕秀琴,武耀康,林璐,等.枯草芽孢杆菌代谢工程改造的策略与工具[J].生物工程学报,2021,37(5):1619-1636.
	LYU X Q, WU Y K, LIN L, et al.. Strategies and tools for metabolic engineering in Bacillus subtilis [J]. Chin. J. Biotechnol., 2021, 37(5):1619-1636.
21	ALTENBUCHNER J. Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system [J]. Appl. Environ. Microbiol., 2016, 82(17):5421-5427.
22	TOYMENTSEVA A A, ALTENBUCHNER J. New CRISPR-Cas9 vectors for genetic modifications of Bacillus species [J/OL]. FEMS Microbiol. Lett., 2019, 366(1):985 [2023-04-23]. .
23	SACHLA A J, ALFONSO A J, HELMANN J D. A simplified method for CRISPR-Cas9 engineering of Bacillus subtilis [J/OL]. Microbiol. Spectr., 2021, 9(2):e00754-21 [2023-04-23]. .
24	YU S, PRICE M A, WANG Y, et al.. CRISPR-dCas9 mediated cytosine deaminase base editing in Bacillus subtilis [J]. ACS Synth. Biol., 2020, 9(7):1781-1789.
25	GARCÍA-MOYANO A, LARSEN Ø, GAYKAWAD S, et al.. Fragment exchange plasmid tools for CRISPR/Cas9-mediated gene integration and protease production in Bacillus subtilis [J/OL]. Appl. Environl. Microb., 2020, 87(1):e02090-20 [2023-04-23]. .
26	HAO W L, SUO F, LIN Q, et al.. Design and construction of portable CRISPR-Cpf1-mediated genome editing in Bacillus subtilis 168 oriented toward multiple utilities [J/OL]. Front Bioeng. and Biotech., 2020, 8:524676 [2023-04-23]. .
27	WU Y K, LIU Y F, LV X Q, et al.. CAMERS-B: CRISPR/Cpf1 assisted multiple-genes editing and regulation system for Bacillus subtilis [J]. Biotechnol. Bioeng., 2020, 117(6):1817-1825.
28	LIM H, CHOI S K. Programmed gRNA removal system for CRISPR-Cas9-mediated multi-round genome editing in Bacillus subtilis [J/OL]. Front Microbiol., 2019, 10:1140 [2023-04-23]. .
29	JAKUTYTE-GIRAITIENE L, GASIUNAS G. Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1 [J]. J. Ind. Microbiol. Biot., 2016, 43(8):1183-1188.
30	WESTBROOK A W, MOO-YOUNG M, CHOU C P. Development of a CRISPR-Cas9 tool kit for comprehensive engineering of Bacillus subtilis [J]. Appl. Environl. Microbiol., 2016, 82(16):4876-4895.
31	ZOU Y, QIU L, XIE A W, et al.. Development and application of a rapid all-in-one plasmid CRISPR-Cas9 system for iterative genome editing in Bacillus subtilis [J/OL]. Microbiol. Cell Fact., 2022, 21(1):173 [2023-04-23]. .
32	ZHANG X Z, YAN X, CUI Z L, et al.. mazF, a novel counter-selectable marker for unmarked chromosomal manipulation in Bacillus subtilis [J/OL]. Nucl. Acids Res., 2016, 34(9):e71 [2023-04-23]. .
33	INÁCIO J M, COSTA C, DE SÁ‍-NOGUEIRA I. Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis [J]. Microbiology, 2003, 149(9):2345-2355.
34	JIANG W Y, BIKARD D, COX D, et al.. CRISPR assisted editing of bacterial genomes [J]. Nat. Biotechnol., 2013, 31(3):233-239.
35	GASIUNAS G, BARRANGOU R, HORVATH P, et al.. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria [J]. Proc. Natl. Acad. Sci. USA, 2012, 109(39):2579-2586.
36	HIRANO H, GOOTENBERG J S, HORII T, et al.. Structure and engineering of Francisella novicida Cas9 [J]. Cell, 2016, 164(5):950-961.
37	KLEINSTIVER B P, PATTANAYAK V, PREW M S, et al.. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects [J]. Nature, 2016, 529(7587):490-495.
38	KLEINSTIVER B P, PREW M S, TSAI S Q, et al.. Engineered CRISPR-Cas9 nucleases with altered PAM specificities [J]. Nature, 2015, 23(7561):481-485.
39	SIKSNYS V, GASIUNAS G. Rewiring Cas9 to target new PAM sequences [J]. Mol. Cell, 2016, 61(6):793-794.
40	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.
41	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.
42	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.
43	RAN F A, HSU P D, LIN C Y, et al.. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity [J]. Cell, 2013, 154(6):1380-1389.
44	STERN A, KEREN L, WURTZEL O, et al.. Self-targeting by CRISPR: gene regulation or autoimmunity? [J]. Trends Genet., 2010, 26(8):335-340.
45	GOMAA A A, KLUMPE H E, LUO M L, et al.. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems [J/OL]. MBio, 2014, 5(1):e00928-13 [2023-04-23]. .
46	VERCOE R B, CHANG J T, DY R L, et al.. Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands [J/OL]. PLoS Genet., 2013, 9(4):e1003454 [2023-04-23]. .
47	HALE C R, MAJUMDAR S, ELMORE J, et al.. Essential features and rational design of CRISPR RNAs that function with the Cas RAMP module complex to cleave RNAs [J]. Mol. Cell, 2012, 45(3):292-302.
48	ZHANG J, ZONG W M, HONG W, et al.. Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for highlevel butanol production [J]. Metab. Eng., 2018, 47:49-59.
49	CASAS-MOLLANO J A, ZINSELMEIER M H, ERICKSON S E, et al.. CRISPR-Cas activators for engineering gene expression in higher eukaryotes [J]. CRISPR J., 2020, 3(5):350-364.
50	WALTON R T, CHRISTIE K A, WHITTAKER M N, et al.. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants [J]. Science, 2020, 368(6488):290-296.
51	KLANSCHNIG M, CSERJAN-PUSCHMANN M, STRIEDNER G, et al.. CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli [J]. Nucleic Acids Res., 2022, 50(18):10772-10784.
52	RONDA C, PEDERSEN L E, SOMMER M O, et al.. CRMAGE: CRISPR optimized MAGE recombineering [J/OL]. Sci. Rep., 2016, 6:19452 [2023-04-23]. .
53	GARST A D, BASSALO M C, PINES G, et al.. Genome-wide mapping of mutations at singlenucleotide resolution for protein, metabolic and genome engineering [J]. Nat. Biotechnol., 2017, 35(1):48-55.
54	LIANG L Y, LIU R M, GARST A D, et al.. CRISPR EnAbled Trackable genome Engineering for isopropanol production in Escherichia coli [J]. Metab. Eng., 2017, 41(3):1-10.
55	BAO Z H, HAMEDIRAD M, XUE P, et al.. Genome-scale engineering of Saccharomyces cerevisiae with single-nucleotide precision [J]. Nat. Biotechnol., 2018, 36(6):505-508.
56	CALLIAS D, VIALETTO E, YU J, et al.. Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria [J/OL]. Nat. Commun., 2023, 14(1):680 [2023-04-23]. .
57	YAN X, YU H J, HONG Q, et al.. Cre/lox system and PCR-based genome engineering in Bacillus subtilis [J]. Appl. Environ. Microbiol., 2008, 74(17):5556-5562.
58	CAI M Z, CHEN P T. Novel combined Cre-Cas system for improved chromosome editing in Bacillus subtilis [J]. J. Biosci. Bioeng., 2021, 132(2):113-119.

分类 Classification	Cas	应用效果 Application	参考文献 Reference
单质粒系统 Single-plasmid	Cas9	25.1 kb片段敲除（89%）以及4.1 kb片段敲除（97%） 25.1 kb fragment knockout （89%） and 4.1 kb fragment knockout （97%）	［21］
	Cas9	单基因编辑（97%） Single gene editing（97%）	［22］
	Cas9	单基因编辑（89%） Single gene editing（89%）	［23］
	dCas9	单基因编辑（Cas9有3个突变位点时效率100%，Cas9有4个突变位点时效率50%） Single gene editing （The efficiency is 100% when Cas9 has 3 mutation sites， and 50% when Cas9 has 4 mutation sites）	［24］
	Cas9	单基因编辑（97%） Single gene editing （97%）	［25］
	Cpf1	单基因缺失（2 kb片段缺失率100%） Single gene deletion （2 kb fragment deletion rate 100%）	［26］
双质粒系统 Double-plasmid	Cas9	单基因插入（100%） Single gene insertion （100%）	［23］
	Cpf1	双基因敲除、多点突变或单基因插入（100%） Double gene knockout， multipoint mutation， or single gene insertion （100%）	［27］
	Cas9	在多轮枯草芽孢杆菌基因编辑中，可连续删除8个胞外蛋白酶基因（每个基因的编辑效率≥90%） In multiple rounds of gene editing for B. subtilis， 8 extracellular protease genes can be continuously deleted （editing efficiency of each gene≥90%）	［28］
	Cpf1	单基因缺失（240 bp片段缺失率100%） Single gene deletion （240 bp fragment deletion rate 100%）	［26］
基因组整合型 Chromosome maintenance	Cas9	预防噬菌体SPP1的感染 Prevention of phage SPP1 infection	［29］
	Cas9	单基因敲除（100%）、双基因敲除（85%）及单基因插入（69%） Single gene knockout （100%）， double gene knockout （85%）， and single gene insertion （69%）	［30］
	Cpf1	单基因敲除（100%）、双基因敲除（58%）及单基因插入（82%） Single gene knockout （100%）， double gene knockout （58%）， and single gene insertion （82%）	［26］

分类 Classification	Cas	应用效果 Application	参考文献 Reference
单质粒系统 Single-plasmid	Cas9	25.1 kb片段敲除（89%）以及4.1 kb片段敲除（97%） 25.1 kb fragment knockout （89%） and 4.1 kb fragment knockout （97%）	［21］
	Cas9	单基因编辑（97%） Single gene editing（97%）	［22］
	Cas9	单基因编辑（89%） Single gene editing（89%）	［23］
	dCas9	单基因编辑（Cas9有3个突变位点时效率100%，Cas9有4个突变位点时效率50%） Single gene editing （The efficiency is 100% when Cas9 has 3 mutation sites， and 50% when Cas9 has 4 mutation sites）	［24］
	Cas9	单基因编辑（97%） Single gene editing （97%）	［25］
	Cpf1	单基因缺失（2 kb片段缺失率100%） Single gene deletion （2 kb fragment deletion rate 100%）	［26］
双质粒系统 Double-plasmid	Cas9	单基因插入（100%） Single gene insertion （100%）	［23］
	Cpf1	双基因敲除、多点突变或单基因插入（100%） Double gene knockout， multipoint mutation， or single gene insertion （100%）	［27］
	Cas9	在多轮枯草芽孢杆菌基因编辑中，可连续删除8个胞外蛋白酶基因（每个基因的编辑效率≥90%） In multiple rounds of gene editing for B. subtilis， 8 extracellular protease genes can be continuously deleted （editing efficiency of each gene≥90%）	［28］
	Cpf1	单基因缺失（240 bp片段缺失率100%） Single gene deletion （240 bp fragment deletion rate 100%）	［26］
基因组整合型 Chromosome maintenance	Cas9	预防噬菌体SPP1的感染 Prevention of phage SPP1 infection	［29］
	Cas9	单基因敲除（100%）、双基因敲除（85%）及单基因插入（69%） Single gene knockout （100%）， double gene knockout （85%）， and single gene insertion （69%）	［30］
	Cpf1	单基因敲除（100%）、双基因敲除（58%）及单基因插入（82%） Single gene knockout （100%）， double gene knockout （58%）， and single gene insertion （82%）	［26］

[1]	李紫馨, 白鸿飞, 谢勇, 李珣, 白立景. 基于CRISPR-Cas9技术建立CSE1L基因敲除C2C12细胞[J]. 中国农业科技导报, 2024, 26(11): 225-233.
[2]	贺春萍, 樊兰艳, 吴贺, 梁艳琼, 吴伟怀, 李锐, 郑服丛. 枯草芽孢杆菌Czk1脂肽物质对橡胶树炭疽病和白粉病的抑制效果研究[J]. 中国农业科技导报, 2023, 25(6): 126-134.
[3]	牛营超, 王星, 郭青云, 戴小华, 袁小勇, 陈琳. 棉花立枯病拮抗细菌的分离鉴定及抑菌活性[J]. 中国农业科技导报, 2023, 25(12): 138-144.
[4]	王强州, 潘诗雨, 方梦雅, 李微, 王佳兴, 刘茵茵, 陈世豪. 基于CRISPR-Cas9技术构建TET2基因敲除的鸡胚成纤维细胞系[J]. 中国农业科技导报, 2023, 25(11): 227-233.
[5]	郝变青, 马利平, 赵永胜, 石文鑫, 王建雄, 景玉川. BC98-Ⅰ和B96-Ⅱ发酵液对马铃薯的防病促生作用及对土壤酶活性的影响[J]. 中国农业科技导报, 2022, 24(8): 116-123.
[6]	翟利敏, 李文通, 冯政, 李华, 裴杨莉. 基因编辑猪的研究现状[J]. 中国农业科技导报, 2022, 24(8): 25-34.
[7]	汪海, 赖锦盛, 王海洋, 李新海. 作物智能设计育种——自然变异的智能组合和人工变异的智能创制[J]. 中国农业科技导报, 2022, 24(6): 1-8.
[8]	彭田伟, 谢会雅, 李思军, 刘怡轩, 帅开峰, 彭媛媛, 王青, 李迪秦. 复硝酚钠和枯草芽孢杆菌复配对烟苗生长和生理指标的影响[J]. 中国农业科技导报, 2022, 24(4): 154-161.
[9]	梁艳琼, 李锐, 吴伟怀, 习金根, 谭施北, 黄兴, 陆英, 贺春萍, 易克贤. 枯草芽孢杆菌Czk1挥发物混合活性组分对橡胶灵芝菌的抑菌机理[J]. 中国农业科技导报, 2022, 24(2): 152-159.
[10]	陈浩, 周菲, 林拥军. 我国作物新种质创制研究进展[J]. 中国农业科技导报, 2022, 24(12): 112-119.
[11]	庄重, 赵龙, 白皓, 毕瑜林, 黄应权, 陈国宏, 常国斌. CRISPR/Cas9技术在家禽育种方面的应用[J]. 中国农业科技导报, 2022, 24(1): 14-23.
[12]	马小倩, 杨涛, 张全, 张洪亮. 水稻新型育种技术研究现状与展望[J]. 中国农业科技导报, 2022, 24(1): 24-30.
[13]	廖嘉明, §, 李春梅§, 张石虎, 李布野, 欧阳昆唏, 陈晓阳. CRISPR/Cas9基因编辑技术的发展及其在植物中的应用[J]. 中国农业科技导报, 2021, 23(12): 20-28.
[14]	梁艳琼,李锐,吴伟怀,习金根,谭施北,郑金龙,黄兴,陆英,贺春萍,易克贤. Bacillus subtilis Czk1挥发性化合物对橡胶树红根病菌的抑制作用[J]. 中国农业科技导报, 2020, 22(11): 116-123.
[15]	薛满德,龙艳,裴新梧*. 基因编辑技术及其在作物育种中的应用与安全管理[J]. 中国农业科技导报, 2018, 20(9): 12-22.