Recent Advances of CRISPR-Cas System in Genome Editing of Bacillus subtilis

doi:10.13304/j.nykjdb.2023.0260

Abstract

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

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

CLC Number:

Q812

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.

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.

Figures/Tables 2

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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］

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