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System types	Strains	Editing types	Editing efficiency	Purposes	References
dCas9	168	Single plasmid, CBE, point mutation	The fluorescence intensity of GFP in RBS library spaned three orders of magnitude	Improved the growth rate and glycerol utilization efficiency	[38]
Cas9	168	Single plasmid, KO, point mutation	TrpC point mutations 100%; 4.1 kb KO 97%; 25.1 kb KO >90%	The first CRISPR/Cas9 tool in B. subtilis	[39]
dCas9	168	Genome integration, CRISPRi	GFP and RFP suppression intensity 0.01-1.00 times	CRISPRi library validation of essential gene functions	[40]
Cas9	168	Dual plasmids, KO, KI, point mutation	Single gene deletion 100%; 38 kb KO 80%; GFP gene KI 97%	Efficient large gene deletions achieved	[41]
dCas9	SCK6	Dual plasmids, CRISPRi	Protease genes vpr, bpr, and nprB expression decreased by 95%, 78%, and 98%	Dual regulation of inhibition and activation, with a 260-fold increased in expression and a 2.6-fold increased in production of the amylase gene BLA	[43]
dCas9	SCK6	Dual plasmids, CRISPRa	GFP expression increased by 2.4-3.8 times
MAD7	168	Single plasmid, KO	Gfpmut3 KO 100%; amyE KO 93%	Efficient editing ensured by homologous recombination repair	[44]
dMAD7	168	Single plasmid, CRISPRi	GFP suppression 1.3-3.5 times; amyE suppression 2.0-2.4 times	Amylase activity decreased by 99.4%	[44]
dCpf1	168	Genome integration, CRISPRi, CRISPRa	GFP suppression intensity 0.008-0.248 times; 1.8-fold activation of mKate fluorescence	Simultaneous gene activation and inhibition achieved by dCpf1-RemA	[45]
Cpf1	168	Dual plasmids, KO, KI, point mutation	Gene KI 74%-82%; double gene KO 100%; 6 site point mutations 100%	NgAgo nucleases improved the efficiency of homologous recombination; inactivation of protease genes	[45]
Cpf1	168	Single plasmid, KO, KI	Small fragment KO >95%; large fragment >80%; 78 kb KO 16.7%	Higher stability of gene KO with Cpf1 protein than Cas9 protein	[46]
dCas9	168	Single plasmid, CBE, point mutation	3 site 100%, 4 site 50%	The first report of CBE application; 5 nt editing window	[47]
dCas9	168	Single plasmid, CBE, point mutation	Efficiency of mutations in 2, 3, 4, and 5 genes were 100%, 100%, 83.3%, and 75.5%, respectively	Construction of 4 types of base editing tools	[48]
Cas9n	168	Single plasmid, CBE, ABE	Editing efficiency of most sites>60%	Enhanced resistance to lanthionine antibiotics	[49]
Cas9	BsMN0	Single plasmid, KI	Single amyQ gene integration efficiency 97.5%-98.6%	Multi-gene one-step insertion and selection	[51]
dCas9	1A976	Single plasmid, CRISPRi, CRISPRa	GFP fluorescence decreased by 5.02-8.36 times	Increased expression of recombinant proteins	[52]
I型CRISPRi	168	Single plasmid, CRISPRi	GFP fluorescence decreased by 26.84-34.18 times	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	YB886 (168 derivative)	Single plasmid, KO	NT	Enhanced resistance to bacteriophage SPP1	[55]
Cas9	TS01	Single plasmid, KO, KI	KO efficiency >40%; 2 kb KI 5%	Bacteriophage gene editing	[56]
Cas9	ATCC 6051A	Single plasmid, KO (gene damage)	SrfC 284 bp deletion efficiency 9.1%; spoIIA deletion efficiency 36%; double gene deletion efficiency 33%	Inhibition of foam formation and spore production	[57]
Cre-Cas9	DB428	Dual plasmids, KO, KI	Gene KO and KI efficiency >95%	Insertion of no resistant genes, inactivation of protease genes	[59]
Cas9 dCas9	1A751	Genome integration, KO, KI, CRISPRi	Point mutation 85%-100%; double gene KO 36%; 2.9 kb KI 69%	β-galactosidase activity decreased by 8 times	[60]
dCas9	168 1A751	Genome integration, CRISPRi	FtsZ relative suppression efficiency 25%-100%	Hyaluronic acid production increased by 204%	[60]
Cas9	168	Dual plasmids, KO	500 bp KO >80%	Self-targeted removal of plasmids	[61]
Cas9n	168	Dual plasmids, point mutation, KO, KI	8 kb KO-82%; 20.5 kb KO-23.6%; 1-2 kb KI >90%	Riboflavin production increased by 59%	[62]
dCas9	168	Genome integration, CRISPRi	GlcNAc production increased 81.7-131.6 g/L	Dynamic regulation of metabolic flux in response to intracellular GlcN6P concentration	[63]
dCas9	BNY (168 derivative)	Genome integration, CRISPRi	Simultaneously suppressed zwf, pfkA, and glmM intensity 0.01-0.10 times	GlcNAc yield increased by 88.5% with reduced by-products	[63]
dCas9	BY14 (168 derivative)	Genome integration, CRISPRi	Pyk and zwf expression decreased by 13.6 and 12.7 times	Lactose production increased by 59.2%; lactose titer increased by 17.4%	[64]
Cas9n	ATCC6051△5	Single plasmid, CBE	NT	Elimination of resistant genes in plasmids	[65]

System types	Strains	Editing types	Editing efficiency	Purposes	References
dCas9	168	Single plasmid, CBE, point mutation	The fluorescence intensity of GFP in RBS library spaned three orders of magnitude	Improved the growth rate and glycerol utilization efficiency	[38]
Cas9	168	Single plasmid, KO, point mutation	TrpC point mutations 100%; 4.1 kb KO 97%; 25.1 kb KO >90%	The first CRISPR/Cas9 tool in B. subtilis	[39]
dCas9	168	Genome integration, CRISPRi	GFP and RFP suppression intensity 0.01-1.00 times	CRISPRi library validation of essential gene functions	[40]
Cas9	168	Dual plasmids, KO, KI, point mutation	Single gene deletion 100%; 38 kb KO 80%; GFP gene KI 97%	Efficient large gene deletions achieved	[41]
dCas9	SCK6	Dual plasmids, CRISPRi	Protease genes vpr, bpr, and nprB expression decreased by 95%, 78%, and 98%	Dual regulation of inhibition and activation, with a 260-fold increased in expression and a 2.6-fold increased in production of the amylase gene BLA	[43]
dCas9	SCK6	Dual plasmids, CRISPRa	GFP expression increased by 2.4-3.8 times
MAD7	168	Single plasmid, KO	Gfpmut3 KO 100%; amyE KO 93%	Efficient editing ensured by homologous recombination repair	[44]
dMAD7	168	Single plasmid, CRISPRi	GFP suppression 1.3-3.5 times; amyE suppression 2.0-2.4 times	Amylase activity decreased by 99.4%	[44]
dCpf1	168	Genome integration, CRISPRi, CRISPRa	GFP suppression intensity 0.008-0.248 times; 1.8-fold activation of mKate fluorescence	Simultaneous gene activation and inhibition achieved by dCpf1-RemA	[45]
Cpf1	168	Dual plasmids, KO, KI, point mutation	Gene KI 74%-82%; double gene KO 100%; 6 site point mutations 100%	NgAgo nucleases improved the efficiency of homologous recombination; inactivation of protease genes	[45]
Cpf1	168	Single plasmid, KO, KI	Small fragment KO >95%; large fragment >80%; 78 kb KO 16.7%	Higher stability of gene KO with Cpf1 protein than Cas9 protein	[46]
dCas9	168	Single plasmid, CBE, point mutation	3 site 100%, 4 site 50%	The first report of CBE application; 5 nt editing window	[47]
dCas9	168	Single plasmid, CBE, point mutation	Efficiency of mutations in 2, 3, 4, and 5 genes were 100%, 100%, 83.3%, and 75.5%, respectively	Construction of 4 types of base editing tools	[48]
Cas9n	168	Single plasmid, CBE, ABE	Editing efficiency of most sites>60%	Enhanced resistance to lanthionine antibiotics	[49]
Cas9	BsMN0	Single plasmid, KI	Single amyQ gene integration efficiency 97.5%-98.6%	Multi-gene one-step insertion and selection	[51]
dCas9	1A976	Single plasmid, CRISPRi, CRISPRa	GFP fluorescence decreased by 5.02-8.36 times	Increased expression of recombinant proteins	[52]
I型CRISPRi	168	Single plasmid, CRISPRi	GFP fluorescence decreased by 26.84-34.18 times	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	YB886 (168 derivative)	Single plasmid, KO	NT	Enhanced resistance to bacteriophage SPP1	[55]
Cas9	TS01	Single plasmid, KO, KI	KO efficiency >40%; 2 kb KI 5%	Bacteriophage gene editing	[56]
Cas9	ATCC 6051A	Single plasmid, KO (gene damage)	SrfC 284 bp deletion efficiency 9.1%; spoIIA deletion efficiency 36%; double gene deletion efficiency 33%	Inhibition of foam formation and spore production	[57]
Cre-Cas9	DB428	Dual plasmids, KO, KI	Gene KO and KI efficiency >95%	Insertion of no resistant genes, inactivation of protease genes	[59]
Cas9 dCas9	1A751	Genome integration, KO, KI, CRISPRi	Point mutation 85%-100%; double gene KO 36%; 2.9 kb KI 69%	β-galactosidase activity decreased by 8 times	[60]
dCas9	168 1A751	Genome integration, CRISPRi	FtsZ relative suppression efficiency 25%-100%	Hyaluronic acid production increased by 204%	[60]
Cas9	168	Dual plasmids, KO	500 bp KO >80%	Self-targeted removal of plasmids	[61]
Cas9n	168	Dual plasmids, point mutation, KO, KI	8 kb KO-82%; 20.5 kb KO-23.6%; 1-2 kb KI >90%	Riboflavin production increased by 59%	[62]
dCas9	168	Genome integration, CRISPRi	GlcNAc production increased 81.7-131.6 g/L	Dynamic regulation of metabolic flux in response to intracellular GlcN6P concentration	[63]
dCas9	BNY (168 derivative)	Genome integration, CRISPRi	Simultaneously suppressed zwf, pfkA, and glmM intensity 0.01-0.10 times	GlcNAc yield increased by 88.5% with reduced by-products	[63]
dCas9	BY14 (168 derivative)	Genome integration, CRISPRi	Pyk and zwf expression decreased by 13.6 and 12.7 times	Lactose production increased by 59.2%; lactose titer increased by 17.4%	[64]
Cas9n	ATCC6051△5	Single plasmid, CBE	NT	Elimination of resistant genes in plasmids	[65]

System types	Strains	Optimization strategy	Optimization results	References
CRISPRa	SCK6	Inhibited and activated dual regulation	The amylase yield increased by 260 times	[43]
CRISPRi	168	Integration of temperature sensor with CRISPRi system	The production of 2′-fucosyl lactose reached 1 839.7 mg/L	[50]
CRISPRi	168	Reconstruction of type I CRISPRi system to control pdhA expression	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	168	Heterologous expression of SeHAS gene, overexpression of pgsA and clsA genes, downregulation of ftsZ gene expression	The hyaluronic acid titer increased by 204%	[54]
CRISPRi	BNY	Inhibition of zwf, pfkA, and glmM genes	The production of N-acetylglucosamine reached 20.5 g/L	[63]
AsCpf1	168	Knockout of eps gene cluster, knockin of tuaD gene at mpr and epr sites	The hyaluronic acid production reached 1.39 g/L	[66]
Cas9	168	Alleviation of transcriptional riboswitch restrictions on riboflavin biosynthesis pathway	The riboflavin production increased by 53%	[67]
Cas9n	168	Regulation of ribB, ribA, and ribH genes	The riboflavin production increased by 59% to reach 1.39 g/L	[68]
dCas12a	S5	Construction of genetic ScrABBLE system	The titer of N-acetylglucosamine reached 183.9 g/L	[69]
Cas9	RLI2019	Knockout of 10 antibiotic resistance genes	The strain was more sensitive to antibiotics	[70-71]
Cas9	RLI2019	Simultaneous integration of eglS, Cel48S, and bglS genes at aprE, epr, and amyE sites	The activities of endoglucanase, exoglucanase, and β-glucosidase were 3.1 times, 6.6 times, and 3.0 times of the starting strain, respectively	[72-74]
Cas9	168	Regulation of key genes in the synthetic pathway trpC2	The lycopene production reached 1.12 mg/L	[75]

System types	Strains	Optimization strategy	Optimization results	References
CRISPRa	SCK6	Inhibited and activated dual regulation	The amylase yield increased by 260 times	[43]
CRISPRi	168	Integration of temperature sensor with CRISPRi system	The production of 2′-fucosyl lactose reached 1 839.7 mg/L	[50]
CRISPRi	168	Reconstruction of type I CRISPRi system to control pdhA expression	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	168	Heterologous expression of SeHAS gene, overexpression of pgsA and clsA genes, downregulation of ftsZ gene expression	The hyaluronic acid titer increased by 204%	[54]
CRISPRi	BNY	Inhibition of zwf, pfkA, and glmM genes	The production of N-acetylglucosamine reached 20.5 g/L	[63]
AsCpf1	168	Knockout of eps gene cluster, knockin of tuaD gene at mpr and epr sites	The hyaluronic acid production reached 1.39 g/L	[66]
Cas9	168	Alleviation of transcriptional riboswitch restrictions on riboflavin biosynthesis pathway	The riboflavin production increased by 53%	[67]
Cas9n	168	Regulation of ribB, ribA, and ribH genes	The riboflavin production increased by 59% to reach 1.39 g/L	[68]
dCas12a	S5	Construction of genetic ScrABBLE system	The titer of N-acetylglucosamine reached 183.9 g/L	[69]
Cas9	RLI2019	Knockout of 10 antibiotic resistance genes	The strain was more sensitive to antibiotics	[70-71]
Cas9	RLI2019	Simultaneous integration of eglS, Cel48S, and bglS genes at aprE, epr, and amyE sites	The activities of endoglucanase, exoglucanase, and β-glucosidase were 3.1 times, 6.6 times, and 3.0 times of the starting strain, respectively	[72-74]
Cas9	168	Regulation of key genes in the synthetic pathway trpC2	The lycopene production reached 1.12 mg/L	[75]

CRISPR系统在枯草芽孢杆菌基因编辑中的研究进展

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公涵萱 ¹^,² , 王智伟 ¹ , 陈玉林 ¹ , 杨雨鑫 ¹ , 刘功炜 ¹

微生物学报 | 综述 2025,65(5): 1867-1884

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微生物学报 | 综述 2025, 65(5): 1867-1884

CRISPR系统在枯草芽孢杆菌基因编辑中的研究进展

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公涵萱¹^,², 王智伟¹, 陈玉林¹, 杨雨鑫¹, 刘功炜¹

作者信息

^1.西北农林科技大学动物科技学院，陕西杨凌

^2.辽宁省微生物科学研究院，辽宁朝阳

Research advances in CRISPR-based genetic editing of Bacillus subtilis

Hanxuan GONG¹^,², Zhiwei WANG¹, Yulin CHEN¹, Yuxin YANG¹, Gongwei LIU¹

Affiliations

^1.College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China

^2.Microbial Research Institute of Liaoning Province, Chaoyang, Liaoning, China

出版时间: 2025-05-04 doi: 10.13343/j.cnki.wsxb.20240688

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摘要

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枯草芽孢杆菌(Bacillus subtilis)是一种公认安全的(generally recognized as safe, GRAS)益生菌，同时也是优良的工业生产底盘菌株，具有异源蛋白分泌能力强、低品质碳源生长特性良好以及无明显密码子偏好性等优点。自2016年以来，成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)基因编辑技术已成功应用于枯草芽孢杆菌，实现了精准的基因点突变、基因敲除、外源基因插入、基因表达调控及碱基替换等多种基因工程操作。这些进展极大地推动了枯草芽孢杆菌作为高效微生物细胞工厂的发展，并在农业、医药、食品以及合成生物学等领域展现了广泛的应用潜力。本文系统回顾了CRISPR系统在枯草芽孢杆菌中的发展历程，重点总结了其在高效生产不同产物方面的应用成果。旨在通过CRISPR系统靶向优化枯草芽孢杆菌的代谢通路，为在工业化生产中高效、稳定地合成目标产物提供参考，并为新型基因编辑系统的进一步开发与应用提供启示。

关键词

枯草芽孢杆菌 / CRISPR / 基因编辑 / 基因表达调控 / 合成生物学

Abstract

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Bacillus subtilis is a generally recognized as safe (GRAS) probiotic and an excellent industrial chassis strain. It possesses advantages such as strong heterologous protein secretion capability, robust growth with low-quality carbon sources, and negligible codon bias. Since 2016, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has been successfully applied to B. subtilis, enabling precise genetic modifications, including point mutations, gene knockout, foreign gene insertion, gene expression regulation, and base editing. These advancements have significantly promoted the development of B. subtilis as an efficient microbial cell factory and have shown broad application potential in agriculture, pharmaceuticals, food production, and synthetic biology. This paper systematically review the development of the CRISPR system in B. subtilis and summarize its application in the efficient production of various products. The aim is to provide insights into the targeted optimization of metabolic pathways in B. subtilisvia the CRISPR system to achieve efficient and stable industrial production of target products, as well as to offer references for the further development and application of novel gene editing systems.

Key words

Bacillus subtilis / CRISPR / gene editing / gene expression regulation / synthetic biology

引用本文

公涵萱, 王智伟, 陈玉林, 杨雨鑫, 刘功炜. CRISPR系统在枯草芽孢杆菌基因编辑中的研究进展. 微生物学报, 2025 , 65 (5) : 1867 -1884 . DOI: 10.13343/j.cnki.wsxb.20240688

Hanxuan GONG, Zhiwei WANG, Yulin CHEN, Yuxin YANG, Gongwei LIU. Research advances in CRISPR-based genetic editing of Bacillus subtilis[J]. Acta Microbiologica Sinica, 2025 , 65 (5) : 1867 -1884 . DOI: 10.13343/j.cnki.wsxb.20240688

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人类生活与微生物活动密切相关。随着系统生物学、合成生物学和进化工程等现代生物技术的快速发展，研究人员能够更深入地理解微生物活动的内在功能机制，对微生物的研究应用已从天然可培养微生物的分离鉴定转向可编程的基因工程菌研究。人类应用微生物至今，经历了3个完全不同的时代：微生物应用1.0时代是充分发挥微生物的自然特性；2.0时代是基于重组DNA技术或物理、化学诱变获得性能更佳的突变菌；3.0时代则是基于现代生物技术的“设计(design)-构建(construction)-评估(evaluation)-优化(optimization) (DCEO)”或“设计(design)-构建(build)-测试(test)-学习(learn) (DBTL)”循环获得基因工程菌，甚至完全从头合成细菌基因组^[1-2]。目前，以大肠埃希氏菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、链霉菌(Streptomyces sp.)等为底盘菌株的基因工程菌在活性化合物生物合成方面取得了快速发展。

枯草芽孢杆菌(Bacillus subtilis)是一种常见的革兰氏阳性菌，也是公认安全的(generally recognized as safe, GRAS)益生菌，具有异源蛋白分泌能力强、在低品质碳源上生长特性良好、无明显密码子偏好性等特点^[3]。在畜禽养殖业、食品加工业、合成生物学等多个领域中应用广泛。在枯草芽孢杆菌中，已经创建了基因蛋白功能数据库SubtiWiki^[4]、启动子和核糖体结合位点(ribosomebinding site, RBS)数据库BioBrick Box^[5]、转录系统数据库DBTBS^[6]以及代谢途径数据库MetaCyc^[7]，这些数据库提供了关于基因功能、代谢途径等方面的关键遗传信息，对开展枯草芽孢杆菌的基因工程改造具有重要的参考意义。此外，与真核表达系统相比，枯草芽孢杆菌易于培养且具有较高的生长速率；与革兰氏阴性细菌表达系统相比，枯草芽孢杆菌表达可有效避免胞内蛋白质积累和不溶性蛋白质包涵体形成的毒性^[8]。通过基因工程技术进行定向遗传改造，枯草芽孢杆菌已应用于酶、维生素、功能性糖等产物合成的微生物细胞工厂构建^[9]。在枯草芽孢杆菌中建立了自发整合、反向选择标记、位点特异性重组等成熟的基因编辑系统。自发整合利用枯草芽孢杆菌能够天然形成感受态的能力，吸收异源DNA分子整合至染色体中，但往往效率低下且具有随机性^[10]。反向选择标记系统通常由靶向染色体基因座侧翼的2个同源区、抗性基因、诱导型启动子控制下的毒素基因和2个直接重复序列组成，常用的反向选择标记包括upp、blaI、mazF、ysbC、hewl和cat等^[3]。位点特异性重组系统引入了重组酶，相比反向选择标记的天然重组，其效率要高得多，常用的位点特异性重组系统包括Xer/dif^[11]和Cre/loxP系统^[12]。

尽管传统基因工程操作方法经过多年发展取得了许多突破，但依然存在技术难度大、费时、成本高等不足，这也限制了枯草芽孢杆菌基因工程改造的进一步发展，尤其是在进行全局代谢网络调控中的应用。自2013年以来，成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其关联(CRISPR associated, Cas)系统(CRISPR/Cas)因操作简单、适用范围广、编辑效率高等诸多优点，迅速发展成为第三代基因编辑技术^[13]。1987年，研究人员首次在大肠埃希氏菌的iap基因内观察到一段特殊重复序列，但当时并未明确其功能^[14]。2002年，这些序列被正式命名为CRISPR序列^[15]。2005年，有研究提出CRISPR识别特定的病毒基因序列，参与细菌免疫机制的假设^[16]。2007年，研究证明了CRISPR/Cas系统确实能够介导原核生物对噬菌体的适应性免疫^[17]。2011年，验证了反式激活CRISPR RNA (trans-activating crRNA, tracrRNA)和CRISPR RNA (crRNA)形成复合物后能够引导Cas9蛋白切割双链^[18]。2012年，研究表明RuvC和HNH结构域对Cas9蛋白发挥内切核酸酶活性至关重要，并且提出了以合成小向导RNA (single guide RNA, sgRNA)代替tracrRNA和crRNA复合物的靶向作用^[19]。2013年，有研究首次报道了CRISPR/Cas9在真核生物基因编辑中的应用^[13]，实现了多基因的高效率敲除，这为后续CRISPR/Cas系统及其衍生技术广泛应用于真核生物和原核生物的靶向基因编辑奠定了基础。2020年，CRISPR/Cas9基因修饰方法的发明者埃马纽埃尔·卡彭蒂耶(Emmanuelle Charpentier)与美国科学家詹妮弗·杜德纳(Jennifer A. Doudna)被授予“诺贝尔化学奖”，这无疑再次掀起了基因编辑领域的新技术革命。

本文系统地介绍了CRISPR/Cas系统的作用原理，梳理了CRISPR基因编辑工具在枯草芽孢杆菌中的发展历程，总结了CRISPR系统靶向编辑枯草芽孢杆菌以促进核黄素、透明质酸、酶蛋白等产物高效生物合成的成功案例，并对CRISPR系统在枯草芽孢杆菌中的发展应用进行了展望，以期为基于全局代谢网络调控的枯草芽孢杆菌基因工程提供参考。

1 CRISPR/Cas系统的作用原理及发展

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1.1 CRISPR/Cas系统的基因编辑原理

CRISPR/Cas系统作为原核生物的获得性免疫系统，主要由CRISPR基因序列与Cas基因组成。CRISPR基因序列能够储存病毒等侵入宿主的核苷酸信息，Cas基因能够编码核酸内切酶等功能性蛋白。CRISPR系统可划分为适应模块、表达模块、干扰模块和辅助模块^[20]。在病毒入侵时，CRISPR系统中的Cas蛋白作为外源性基因的识别者，识别protospacer adjacent motif (PAM)序列并定位入侵的外源遗传元件。经Cas核酸酶精确切割后，外源基因以间隔序列的形式整合至CRISPR序列中。作为前体crRNA生成的模板，间隔序列存储着外源基因的遗传信息。当相同类型的病毒二次入侵时，宿主细胞会启动防御机制，利用转录和复杂的分子机制，重组具备高度特异性识别能力的复合体，基于碱基互补配对原则，识别并切割基因序列，致使双链DNA的特异性断裂，从而有效阻断病毒的复制^[21]。以典型的CRISPR/Cas9系统为例，来源于化脓性链球菌的Cas9蛋白(SpCas9)，包括切割靶向链的HNH和切割非靶向链的RuvC两个核酸酶结构域，其靶向PAM序列为NGG^[22]。经过优化后的CRISPR/Cas9系统仅需sgRNA引导Cas9蛋白在靶基因位点切割DNA双链，之后通过随机突变、同源重组(homologous recombination, HDR)或非同源末端连接(non-homologous end joining, NHEJ)进行断裂的双链修复，修复过程中即可发生基因编辑(图1)^[23]。

1.2 CRISPR/Cas系统的分类

基于Cas蛋白的组成，CRISPR系统被分为两大类。Ⅰ类CRISPR系统(如I型、III型和IV型系统)需要多种Cas蛋白才能发挥作用，而Ⅱ类CRISPR系统(如II型、V型和VI型系统)只需要一个Cas蛋白实现其功能^[23]。Ⅱ类CRISPR系统中的每个亚型使用独特的Cas蛋白发挥切割活性，例如，II型系统为Cas9蛋白；V型系统中包括Cas12a (Cpf1)、Cas12b (C2c1)、Cas12c(C2c3)、Cas12d (CasY)、Cas12e (CasX)、Cas12g、Cas12h和Cas12i；VI型系统则包括Cas13a(C2c2)、Cas13b和Cas13c^[23-24]。xCas9蛋白^[25]、SpRY^[26]等Cas变体蛋白的不断出现，进一步提高了精准基因编辑的可行性。可以预见，随着研究的不断深入，特异性更强、蛋白更小、编辑范围更广、效率更高的Cas蛋白依然会不断被发现并应用于基因编辑研究。

1.3 CRISPR/Cas系统衍生的基因编辑工具发展

Cas9蛋白HNH结构域的H840A以及RuvC结构域的D10A发生点突变获得缺乏内切核酸酶活性的变体蛋白dCas9，但其依然能结合靶位点DNA序列，dCas9融合阻遏因子可实现对靶基因的抑制表达调控，即CRISPR干扰(CRISPR interference, CRISPRi) (图2)^[27]。在细菌的实际应用中，dCas9无需融合阻遏因子同样能有效发挥基因抑制作用，CRISPRi借助sgRNA的靶向作用，只需改变20 nt的靶向碱基即可实现靶基因的切换。此外，dCas9可直接融合激活因子，在sgRNA的引导下实现对靶基因的激活表达调控，即CRISPR激活(CRISPR activation, CRISPRa) (图2)^[19,27]。已报道的激活因子包括α亚基、ω亚基、SoxS、TetD等。与CRISPRi不同的是，CRISPRa的有效靶位点区域在转录起始位点的上游，并且仅在有限的靶区域表现出有效激活作用^[27-28]。

Komor等^[29]利用dCas9结合脱氨酶、逆转录酶等工具，开发出了可实现特定碱基精确点突变的碱基编辑系统(base editor, BE)，包括能进行C到T替换的胞嘧啶碱基编辑系统(cytosine base editor, CBE)，A到G替换的腺嘌呤碱基编辑系统(adenine base editor, ABE)^[30]，以及能进行A、T、C、G 4种碱基任意变化的先导编辑器(prime editing, PE)系统^[31]。针对碱基编辑器编辑方式的单一性，众多研究团队开发出了能进行C到G替换及同时进行C到T与A到G替换的碱基编辑器变体，这些变体的提出进一步扩展了基因编辑的应用方式及范围^[32-33]。BE系统已经在大肠埃希氏菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、谷氨酸棒杆菌(Corynebacterium glutamicum)、肺炎克雷伯氏菌(Klebsiella pneumoniae)等多种细菌中实现了碱基编辑^[34]。

BE和PE系统可实现靶位点的单碱基替换，导致编码基因的提前终止，从而用于基因功能研究或有害基因功能沉默(图3)。在大肠埃希氏菌中，运用BE3系统将CAA、CAG和CGA突变为终止密码子(TAA、TAG和TGA)，实现了100%编辑效率的四环素抗性基因功能丧失，且编辑效率顺序为TC≥CC≥AC≥GC；同时在靶向布鲁氏菌的致病力因子(virB10)时也表现出高效率的基因功能失活^[37]。Wang等^[38]在CBE的基础上开发了一种以碱基编辑为目标和无模板的基因表达调控(base editor-targeted and template-free expression regulation, BETTER)方法，用于优化谷氨酸棒杆菌的木糖代谢途径。为验证BETTER方法在复杂多基因调控过程中的有效性，Wang等^[38]设计了同时靶向谷氨酸棒杆菌番茄红素生物合成途径中10个关键基因的BETTER工具，获得了番茄红素产量提高4.8倍的工程菌。总的来说，建立基于基因组规模的BE和PE编辑系统、降低脱靶效率、增大编辑通量等方面都是必要的。

2 枯草芽孢杆菌中CRISPR/Cas基因编辑工具的发展

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自2016年以来，CRISPR/Cas系统在枯草芽孢杆菌中实现了高效率的单基因点突变、多基因点突变、单基因敲除、多基因缺失、大片段删除和外源基因插入等基因编辑操作，同时，CRISPRi、CRISPRa和BE系统等遗传工具的开发应用，以及将CRISPR系统结合生物传感器实现代谢途径多模块的动态调控，已拓展到生物制造领域。2016年，CRISPR/Cas系统首次应用于枯草芽孢杆菌的遗传改造，通过CRISPR/Cas9技术成功修复了B. subtilis 168的trpC2基因突变^[39]。同时，以dCas9为基础的CRISPRi也应用于枯草芽孢杆菌全基因组规模的基因功能研究^[40]。2017年，基于CRISPR/Cas9技术，在枯草芽孢杆菌中实现了高效率的大片段基因组缺失和基因敲入^[41]。2018年，CRISPR/Cas9n应用于枯草芽孢杆菌^[42]。2019年，Lu等^[43]开发了基于dCas9的CRISPRa工具。2020年，CRISPR/Cpf1系统和MAD7变体用于枯草芽孢杆菌基因编辑^[44-46]；CBE首次应用于枯草芽孢杆菌^[47]。2021年，Kim等^[48]构建了融合rAPOBEC1和PmCDA1的4种CBE；BETTER方法也应用到B. subtilis 168中构建RBS文库^[38]。2022年，ABE结合CBE应用于枯草芽孢杆菌定向进化，成功增强了枯草芽孢杆菌对羊毛硫细菌素的抗性^[49]，温度传感器与CRISPRi系统结合应用于枯草芽孢杆菌，成功提高了枯草芽孢杆菌的2′-岩藻糖基乳糖产量^[50]。2023年，Ferrando等^[51]开发了CRISPR系统介导的基因组多基因插入方法，实现了枯草芽孢杆菌中一步多基因插入。2024年，Zhu等^[52]结合全基因组规模的CRISPRi筛选和靶向CRISPRa激活，用于增强枯草芽孢杆菌重组蛋白表达；同时CRISPR的I-E型可编程系统应用于枯草芽孢杆菌^[53]。随着CRISPRi、CRISPRa和BE等CRISPR基因编辑技术的迭代更新，将进一步拓宽枯草芽孢杆菌中的可编程基因操作应用范围，促进其代谢工程研究和合成生物学应用(图4)。

2.1 应用CRISPR/Cas系统实现高效基因敲除和敲入

在枯草芽孢杆菌中，CRISPR/Cas系统已实现高效率的点突变、大片段基因敲除和敲入，并且在增加透明质酸产量^[54]、研究噬菌体生物学特性^[55-56]等方面均有成功应用。2016年，在pJOE8999载体上同时连接了靶向sgRNA和同源修复模板，构建的CRISPR/Cas9系统实现了100%的trpC点突变基因修复、97%的4.1 kb片段敲除和89%的25.1 kb大片段敲除^[39]。该pJOE8999载体在后续多项研究中均成功实现高效率基因敲除或插入^[42,56]，验证了该CRISPR/Cas9系统的稳定性。然而，在另一项研究中，CRISPR/Cas9引起B. subtilis ATCC6051a双基因缺失效率约为33%，6 bp和11 bp基因插入效率约为43%，284 bp基因缺失效率仅为9.1%，且以PCR线性片段提供修复模板时未发生编辑。作者将CRISPR系统的低效率归因于出发菌株为未驯化的野生菌株，转化效率低，不易进行遗传操作^[57]。2016年，发表了枯草芽孢杆菌的CRISPR/Cas9操作指南^[58]，该指南详细说明了试剂组成、处理时间、载体构建、检测方法(随机缺失、插入、点突变)等试验流程，运用该方法验证了MutS2基因缺失导致B. subtilis PY79对天然抗生素丝裂霉素(mitomycin C, MMC)敏感^[58]。然而，该方法需要同时提供tracrRNA和crRNA，在实际应用中较为繁琐。研究发现NgAgo能促进CRISPR/Cpf1系统的同源重组，应用于B. subtilis 168可实现双基因敲除，但对3个基因同时敲除无效；当同源修复模板长度为1 kb时，同时6个位点的点突变和单基因插入效率均达到100%；且以PCR线性化产物和质粒形式提供修复模板均能成功发生预期编辑，大大提高了编辑效率^[45]。Cai等^[59]将CRISPR系统结合重组酶Cre，实现在枯草芽孢杆菌高效无抗性标记的基因插入和敲除(效率>95%)，并且以基因组嵌入的形式同时失活了蛋白酶基因mpr并表征了角蛋白酶功能。Ferrando等^[51]将CRISPR/Cas9系统结合高通量比色筛选方法，1周内完成了最多3个拷贝基因在B. subtilis BsMN0中的同时插入，缩短了工程菌构建周期，快速构建了无质粒、高表达的枯草芽孢杆菌。

由此可见，CRISPR/Cas系统自应用于枯草芽孢杆菌以来，发展出了单质粒系统、双质粒系统和基因组嵌入表达系统，基因编辑效率和稳定性不断提升(表1)，不同系统也表现出明显的优缺点。单质粒系统的质粒转化效率高，能有效减轻宿主菌的负担，提高工程菌存活率；但同源重组效率很大程度上取决于同源臂的长度。双质粒系统的稳定性欠佳，并且可能在宿主菌中引入适应性压力，部分枯草芽孢杆菌难以转化2个质粒，同时质粒抗性选择较为严格，在迭代基因组编辑中需要更多时间进行质粒去除操作。基因组整合系统由于Cas蛋白插入到宿主基因组上，表达更为稳定，但同时由于基因组插入拷贝数低，可能因Cas蛋白表达量不足而引起编辑效率降低。

2.2 CRISPRi和CRISPRa实现多通路基因表达调控

CRISPRi和CRISPRa可特异性抑制或激活基因转录，实现目的基因表达的下调或激活。2016年，CRISPR/Cas9首次应用于枯草芽孢杆菌时，CRISPRi也随即应用到枯草芽孢杆菌中，并在基因功能研究中展现出巨大潜力^[40]。研究人员将木糖诱导启动子驱动的dCas9整合到lacA位点，将组成型启动子P _veg 驱动的sgRNA插入到amyE位点，获得CRISPRi工程菌，并通过红色荧光蛋白(red fluorescent protein, RFP)和绿色荧光蛋白(green fluorescent protein, GFP)验证了CRISPRi对上游基因和下游基因均表现出抑制作用^[40]。Peters等^[40]设计了靶向B. subtilis 168中289个必需基因的sgRNA文库，发现约95%的CRISPRi菌株表现出生长曲线滞后，约80%的菌株最大生长速率与对照相当；CRISPRi必需基因轻度敲减菌株约94%表现出菌落变小，且必需基因敲减菌株超过60%以菌落形态缺陷为最终表型。通过CRISPRi验证必需基因的功能，发现未知功能必需基因ylaN与菌体铁稳态缺陷相关，但当外源添加铁时，ylaN基因则变为非必需^[40]。在以枯草芽孢杆菌为底盘菌株的工业生产中，CRISPRi已用于增加缬氨酸^[60]、透明质酸^[60]、N-乙酰氨基葡萄糖^[63]、淀粉酶^[43]等产物的产量，其本质是通过CRISPRi抑制产物合成竞争途径的基因表达。Westbrook等^[54]利用CRISPRi同时抑制枯草芽孢杆菌的pdhA、leuA、ilvA及sigF基因表达，实现缬氨酸浓度升高14倍以上。最近的一项研究中，通过改造Cas基因mRNA的5′非翻译区，优化了I型CRISPRi系统的功效，并应用于枯草芽孢杆菌生物合成d-泛酸的过程^[53]。

2019年，Lu等^[43]发表了CRISPRa首次应用于枯草芽孢杆菌的研究成果，研究人员将dCas9分别融合激活因子ω和α后，检测到GFP报告基因不同程度地增强表达，且在靶向外源和内源淀粉酶基因时均检测到酶活性的升高；但是激活作用存在高度靶位点依赖性，通过在转录起始位点(transcription start site, TSS)上游位置进行严格的sgRNA设计，dCas9-ω能够以位置依赖性方式同时实现激活和抑制基因表达，这说明CRISPRa能有效激活枯草芽孢杆菌中的内源和外源基因表达。然而，与真核细胞中多个激活因子的叠加募集作用能够进一步增强靶基因表达不同，在枯草芽孢杆菌中观察到多激活因子融合对激活基因表达有负效应^[43]。Wu等^[45]在dCpf1蛋白的C端融合了10种不同激活蛋白，发现dCpf1-RemA具有最佳的激活作用，设计靶向模板链TSS上游60-277 bp的不同crRNA，发现90 bp位置是实现同时抑制和激活的最佳位点，作者借助dCpf1-RemA工具将枯草芽孢杆菌的乙偶姻产量提高了44.8%。尽管CRISPR系统在枯草芽孢杆菌的基因表达调控应用中得到了快速发展，但依然存在一些问题，如针对多基因表达调控的载体构建操作较为繁琐；同时进行CRISPRi和CRISPRa调控时，靶位点的有效作用区域限制比较严格，且针对不同基因的最佳靶向区域变化较大；CRISPRa工具的有效激活调控因子较少，且往往不具有普适性，这些问题都需要进一步地研究解决。

2.3 碱基编辑器的开发应用

2020年，胞嘧啶碱基编辑系统CBE首次应用于枯草芽孢杆菌。研究人员发现，CBE在枯草芽孢杆菌中具有5 nt的编辑窗口范围，在PAM位点上游-16‒-20位置的编辑效率显著不同，以淀粉酶基因amyE为靶位点时，-16‒-20位置的编辑效率分别为2%、51%、100%、20%和13%；当运用CBE工具同时靶向3个位点和4个位点时，编辑效率分别为100%和50%；因此，仅通过两轮基因编辑即可实现8个蛋白酶基因的失活，并检测到蛋白酶活性显著降低至与WB800菌株相当；进一步分析BE系统在B. subtilis 168基因组(共4 106个基因)的可编辑范围发现，共有3 266个非必需基因(占85.1%)可编辑，而其他569个非必需基因的编码区缺乏合适的PAM位点^[47]。后续研究中，研究人员分别将大鼠胞嘧啶脱氨酶(rat cytidine deaminase, rAPOBEC1)和海七鳃鳗胞嘧啶脱氨酶(Petromyzon marinus cytidine deaminase, PmCDA1)融合在dCas9蛋白的N端和C端，并添加不同数量的蛋白降解标签和尿嘧啶糖基化酶抑制剂(uracil glycosylase inhibitor, UGI)，构建了4种不同的CBE：rAPOBEC1-dCas9-UGI (CBE1)、rAPOBEC1-dCas9-UGI-UGI (CBE2)、dCas9-PmCDA1-UGI-LVA (CBE3)和dCas9-PmCDA1-UGI (CBE4)，CBE1-CBE4使GFP失活的效率分别达到97.07%、77.85%、90.17%和98.25%^[48]。分析CBE4的编辑窗口范围发现，在PAM位点上游第17-20位碱基的突变效率超过71%，而在第11-15位的效率低至5%-20%，第1-10位未检测到有效编辑^[48]。同时，研究发现启动子表达强度影响多基因突变效率，CBE4的高表达可能会增加细胞负担从而降低多基因同时编辑效率；当诱导dCas9表达的启动子替换为弱表达强度的启动子后，2、3、4和5个基因同时点突变的效率分别为100%、100%、83.3%和75.5%，但同时也检测到非预期的脱靶效应^[48]。为了验证BETTER方法在不同微生物中进行基因表达调控的普适性，研究人员将其应用到了B. subtilis 168中构建RBS文库，获得GFP表达强度差异跨越3个数量级的文库，验证了BETTER方法在枯草芽孢杆菌中的适用性；并且将来源于克雷伯氏菌的甘油利用途径以串联基因簇(P _gapDH -glpFKs-dhaD-dhaK)形式整合到枯草芽孢杆菌的基因组中，以甘油为唯一碳源，进行连续6代富集培养后，获得了生长速度快、可高效利用甘油的枯草芽孢杆菌^[38]。Chen等^[65]使用CRISPR/Cas9n-AID碱基编辑系统，将碱基C转化为T，将卡那霉素耐药基因的密码子转化为终止密码子，消除枯草芽孢杆菌质粒中的卡那霉素抗性基因，获得的无抗性标记重组菌的γ-谷酰基肽酶产量提高54倍。研究人员通过对CRISPR阵列、nCas9、胞嘧啶和腺苷脱氨酶进行重编程，完成了CBE结合ABE的可编程双碱基编辑器的构建，实现了基因组规模的单个或多个碱基转换，用于底盘细菌的快速进化，并成功增强了枯草芽孢杆菌的抗羊毛硫细菌素能力^[49]。

由于BE系统在实现单碱基的精准替换方面表现出明显优势，后续在枯草芽孢杆菌中的研究应用将着重于文库构建寻找特定表型功能提升的突变株，或者对潜在耐药基因、毒力因子、致病性基因等特定有害基因功能的失活。此外，BE系统在未知基因功能的研究也能发挥重要作用。另一方面，尽管CBE和ABE已经在枯草芽孢杆菌中成功应用，但仍存在可编辑范围较窄的问题，且在同时编辑多个靶基因时，编辑效率明显下降或存在脱靶效应，因此在枯草芽孢杆菌中的多路编辑的BE系统有很大的提升空间。此外，PE系统至今尚未见在枯草芽孢杆菌中的文献报道，未来探究PE系统在枯草芽孢杆菌中的编辑应用也具有重要意义。

3 CRISPR基因编辑技术在枯草芽孢杆菌底盘菌株改造中的应用

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在合成生物学中，进行枯草芽孢杆菌底盘菌株改造的核心思路之一是对目的产物合成竞争途径的抑制以及对合成途径限速酶基因的过表达，从而使更多的代谢通量流入目的产物合成途径，应用CRISPR/Cas系统能方便快捷地达到这一目的。通过CRISPR/Cas系统对枯草芽孢杆菌的基因表达调控和全局代谢网络优化，已成功实现透明质酸、N-乙酰氨基葡萄糖、核黄素等各类活性化合物的分泌表达，以及促进蛋白酶、纤维素酶等生物酶的高效生产^[9]。

Westbrook等^[54]利用CRISPR/Cas9技术异源表达SeHAS基因，过表达pgsA和clsA基因，并降低ftsZ基因的表达量，最终使枯草芽孢杆菌的透明质酸滴度增加了204%。Zhao等^[66]利用CRISPR/AsCpf1系统，在B. subtilis 168中高效完成了透明质酸合成酶基因(hasA)、UDP-葡萄糖-脱氢酶基因(tuaD)在mpr、epr位点的基因敲入以及eps基因簇的敲除，实现了透明质酸的异源生产，产量达到1.39 g/L。

Boumezbeur等^[67]基于CRISPR/Cas9技术降低了转录核糖开关对枯草芽孢杆菌嘌呤和核黄素合成代谢途径的限制，最终使核黄素产量增加了53%。Liu等^[68]利用CRISPR/Cas9n系统调控核黄素操纵子中的ribB、ribA和ribH等3个基因，最终使核黄素产量提高了59%，达到1.39 g/L。

Wu等^[63]通过CRISPRi抑制枯草芽孢杆菌中N-乙酰氨基葡萄糖合成竞争途径的3个基因(zwf、pfkA、glmM)，发现当靶向zwf、pfkA、glmM位点的前端、中端、后端时，抑制强度分别为高、中、低水平，最终获得20.5 g/L的最大N-乙酰氨基葡萄糖产量，显著提升了木糖利用效率。在另一项研究中，Wu等^[69]基于CRISPR-dCas12a系统构建了遗传环路级联ScrABBLE系统，以实现代谢途径的高效多重优化，在生物反应器中，枯草芽孢杆菌N-乙酰氨基葡萄糖的滴度达到183.9 g/L。

在研究枯草芽孢杆菌产纤维素酶特性的工作中，研究人员以白蚁肠道来源的野生型B. subtilis RLI2019为出发菌，以超过80%的效率连续敲除其10个耐药性基因，获得无耐药性基因菌株，该菌株依然保留了高效的纤维素降解能力，并且能够提高动物生长性能^[70-71]。同时，通过CRISPR/Cas9技术，以基因组多位点整合的方式在B. subtilis RLI2019的aprE、epr和amyE位点分别敲入eglS、Cel48S和bglS，实现了三元纤维素酶基因的高效重组表达，重组工程菌的内切葡聚糖酶活性、外切葡聚糖酶活性和β-葡萄糖苷酶活性分别是出发菌的3.1倍、6.6倍和3.0倍，显著增强了对农作物秸秆的降解能力^[72-74]。未来，通过CRISPRi结合CRISPRa重定向枯草芽孢杆菌的代谢通量，有望进一步提高其纤维素酶活性，促进枯草芽孢杆菌在木质纤维素生物降解方面的应用。

此外，基于CRISPR基因编辑技术进行枯草芽孢杆菌全局代谢通路优化的遗传改造策略，已成功构建出涵盖酶制剂(淀粉酶^[43])、天然产物(番茄红素^[75])及高附加值营养添加剂(d-泛酸^[53]、2′-岩藻糖基乳糖^[50])等高效合成的微生物细胞工厂(表2)。

4 总结与展望

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遗传改造工具的技术迭代是推动基因工程菌研究的重要基础，而系统代谢工程是构建理想微生物细胞工厂的有力手段。在过去近10年间，以枯草芽孢杆菌为底盘菌的CRISPR基因编辑技术应用已突破传统基因工程的局限。通过目标基因过表达、代谢途径优化及代谢网络重构等策略，显著提升了目的产物的生物合成效率，为工业级量产提供了创新性解决方案。当前研究已形成以模式菌B. subtilis 168及其衍生菌株为核心的技术体系，但仍存在一些关键问题亟待解决，例如特异性不足导致的脱靶效应、全局代谢网络多层级调控的复杂性、自然环境分离的野生型菌株CRISPR基因编辑体系的不成熟性，以及工程菌生物安全性评估体系的不完善性等。在未来的技术发展中，应重点关注以下几个方面：(1) 全局基因表达调控系统的开发，例如开发CRISPR系统与群体感应系统耦合的自适应调控平台，实现代谢通路的动态平衡；(2) CRISPR基因编辑工具的普适性应用，将经过模式菌验证的CRISPR工具包拓展至野生菌株；(3) 在提高CRISPR基因编辑精确性方面，须持续致力于发掘毒性低、分子量小、靶向范围宽、编辑效率高的新型核酸酶，同时，整合深度学习算法，甚至引入人工智能技术，构建脱靶效应预测模型。

为实现工程菌最终的工业化安全应用，全面评估基于CRISPR系统重构的工程菌遗传稳定性和潜在风险，并制定相应的安全性检测标准和风险调控策略至关重要。总体而言，随着CRISPR基因编辑技术的持续发展，枯草芽孢杆菌中的可编程基因操作应用前景将更为广阔，并将进一步推动其在生物制造和合成生物学领域的发展应用。

基金

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国家重点研发计划(2022YFD1300201)
中国博士后科学基金(2023M732893)
陕西省博士后科研项目(2023BSHEDZZ145)
国家绒毛用羊产业技术体系(CARS-39-12)

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2025年第65卷第5期

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doi: 10.13343/j.cnki.wsxb.20240688

接收时间：2024-11-05
首发时间：2026-02-05
出版时间：2025-05-04

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收稿日期：2024-11-05
录用日期：2025-02-25

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National Key Research and Development Program of China(2022YFD1300201)

国家重点研发计划(2022YFD1300201)

China Postdoctoral Science Foundation(2023M732893)

中国博士后科学基金(2023M732893)

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陕西省博士后科研项目(2023BSHEDZZ145)

China Agriculture Research System(CARS-39-12)

国家绒毛用羊产业技术体系(CARS-39-12)

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^1.西北农林科技大学动物科技学院，陕西杨凌

^2.辽宁省微生物科学研究院，辽宁朝阳

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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System types	Strains	Editing types	Editing efficiency	Purposes	References
dCas9	168	Single plasmid, CBE, point mutation	The fluorescence intensity of GFP in RBS library spaned three orders of magnitude	Improved the growth rate and glycerol utilization efficiency	[38]
Cas9	168	Single plasmid, KO, point mutation	TrpC point mutations 100%; 4.1 kb KO 97%; 25.1 kb KO >90%	The first CRISPR/Cas9 tool in B. subtilis	[39]
dCas9	168	Genome integration, CRISPRi	GFP and RFP suppression intensity 0.01-1.00 times	CRISPRi library validation of essential gene functions	[40]
Cas9	168	Dual plasmids, KO, KI, point mutation	Single gene deletion 100%; 38 kb KO 80%; GFP gene KI 97%	Efficient large gene deletions achieved	[41]
dCas9	SCK6	Dual plasmids, CRISPRi	Protease genes vpr, bpr, and nprB expression decreased by 95%, 78%, and 98%	Dual regulation of inhibition and activation, with a 260-fold increased in expression and a 2.6-fold increased in production of the amylase gene BLA	[43]
dCas9	SCK6	Dual plasmids, CRISPRa	GFP expression increased by 2.4-3.8 times
MAD7	168	Single plasmid, KO	Gfpmut3 KO 100%; amyE KO 93%	Efficient editing ensured by homologous recombination repair	[44]
dMAD7	168	Single plasmid, CRISPRi	GFP suppression 1.3-3.5 times; amyE suppression 2.0-2.4 times	Amylase activity decreased by 99.4%	[44]
dCpf1	168	Genome integration, CRISPRi, CRISPRa	GFP suppression intensity 0.008-0.248 times; 1.8-fold activation of mKate fluorescence	Simultaneous gene activation and inhibition achieved by dCpf1-RemA	[45]
Cpf1	168	Dual plasmids, KO, KI, point mutation	Gene KI 74%-82%; double gene KO 100%; 6 site point mutations 100%	NgAgo nucleases improved the efficiency of homologous recombination; inactivation of protease genes	[45]
Cpf1	168	Single plasmid, KO, KI	Small fragment KO >95%; large fragment >80%; 78 kb KO 16.7%	Higher stability of gene KO with Cpf1 protein than Cas9 protein	[46]
dCas9	168	Single plasmid, CBE, point mutation	3 site 100%, 4 site 50%	The first report of CBE application; 5 nt editing window	[47]
dCas9	168	Single plasmid, CBE, point mutation	Efficiency of mutations in 2, 3, 4, and 5 genes were 100%, 100%, 83.3%, and 75.5%, respectively	Construction of 4 types of base editing tools	[48]
Cas9n	168	Single plasmid, CBE, ABE	Editing efficiency of most sites>60%	Enhanced resistance to lanthionine antibiotics	[49]
Cas9	BsMN0	Single plasmid, KI	Single amyQ gene integration efficiency 97.5%-98.6%	Multi-gene one-step insertion and selection	[51]
dCas9	1A976	Single plasmid, CRISPRi, CRISPRa	GFP fluorescence decreased by 5.02-8.36 times	Increased expression of recombinant proteins	[52]
I型CRISPRi	168	Single plasmid, CRISPRi	GFP fluorescence decreased by 26.84-34.18 times	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	YB886 (168 derivative)	Single plasmid, KO	NT	Enhanced resistance to bacteriophage SPP1	[55]
Cas9	TS01	Single plasmid, KO, KI	KO efficiency >40%; 2 kb KI 5%	Bacteriophage gene editing	[56]
Cas9	ATCC 6051A	Single plasmid, KO (gene damage)	SrfC 284 bp deletion efficiency 9.1%; spoIIA deletion efficiency 36%; double gene deletion efficiency 33%	Inhibition of foam formation and spore production	[57]
Cre-Cas9	DB428	Dual plasmids, KO, KI	Gene KO and KI efficiency >95%	Insertion of no resistant genes, inactivation of protease genes	[59]
Cas9 dCas9	1A751	Genome integration, KO, KI, CRISPRi	Point mutation 85%-100%; double gene KO 36%; 2.9 kb KI 69%	β-galactosidase activity decreased by 8 times	[60]
dCas9	168 1A751	Genome integration, CRISPRi	FtsZ relative suppression efficiency 25%-100%	Hyaluronic acid production increased by 204%	[60]
Cas9	168	Dual plasmids, KO	500 bp KO >80%	Self-targeted removal of plasmids	[61]
Cas9n	168	Dual plasmids, point mutation, KO, KI	8 kb KO-82%; 20.5 kb KO-23.6%; 1-2 kb KI >90%	Riboflavin production increased by 59%	[62]
dCas9	168	Genome integration, CRISPRi	GlcNAc production increased 81.7-131.6 g/L	Dynamic regulation of metabolic flux in response to intracellular GlcN6P concentration	[63]
dCas9	BNY (168 derivative)	Genome integration, CRISPRi	Simultaneously suppressed zwf, pfkA, and glmM intensity 0.01-0.10 times	GlcNAc yield increased by 88.5% with reduced by-products	[63]
dCas9	BY14 (168 derivative)	Genome integration, CRISPRi	Pyk and zwf expression decreased by 13.6 and 12.7 times	Lactose production increased by 59.2%; lactose titer increased by 17.4%	[64]
Cas9n	ATCC6051△5	Single plasmid, CBE	NT	Elimination of resistant genes in plasmids	[65]

System types

Strains

Editing types

Editing efficiency

Purposes

References

dCas9

168

Single plasmid, CBE, point mutation

The fluorescence intensity of GFP in RBS library spaned three orders of magnitude

Improved the growth rate and glycerol utilization efficiency

[38]

Cas9

168

Single plasmid, KO, point mutation

TrpC point mutations 100%; 4.1 kb KO 97%; 25.1 kb KO >90%

The first CRISPR/Cas9 tool in B. subtilis

[39]

dCas9

168

Genome integration, CRISPRi

GFP and RFP suppression intensity 0.01-1.00 times

CRISPRi library validation of essential gene functions

[40]

Cas9

168

Dual plasmids, KO, KI, point mutation

Single gene deletion 100%; 38 kb KO 80%; GFP gene KI 97%

Efficient large gene deletions achieved

[41]

dCas9

SCK6

Dual plasmids, CRISPRi

Protease genes vpr, bpr, and nprB expression decreased by 95%, 78%, and 98%

Dual regulation of inhibition and activation, with a 260-fold increased in expression and a 2.6-fold increased in production of the amylase gene BLA

[43]

dCas9

SCK6

Dual plasmids, CRISPRa

GFP expression increased by 2.4-3.8 times

MAD7

168

Single plasmid, KO

Gfpmut3 KO 100%; amyE KO 93%

Efficient editing ensured by homologous recombination repair

[44]

dMAD7

168

Single plasmid, CRISPRi

GFP suppression 1.3-3.5 times; amyE suppression 2.0-2.4 times

Amylase activity decreased by 99.4%

[44]

dCpf1

168

Genome integration, CRISPRi, CRISPRa

GFP suppression intensity 0.008-0.248 times; 1.8-fold activation of mKate fluorescence

Simultaneous gene activation and inhibition achieved by dCpf1-RemA

[45]

Cpf1

168

Dual plasmids, KO, KI, point mutation

Gene KI 74%-82%; double gene KO 100%; 6 site point mutations 100%

NgAgo nucleases improved the efficiency of homologous recombination;

inactivation of protease genes

[45]

Cpf1

168

Single plasmid, KO, KI

Small fragment KO >95%; large fragment >80%; 78 kb KO 16.7%

Higher stability of gene KO with Cpf1 protein than Cas9 protein

[46]

dCas9

168

Single plasmid, CBE, point mutation

3 site 100%, 4 site 50%

The first report of CBE application; 5 nt editing window

[47]

dCas9

168

Single plasmid, CBE, point mutation

Efficiency of mutations in 2, 3, 4, and 5 genes were 100%, 100%, 83.3%, and 75.5%, respectively

Construction of 4 types of base editing tools

[48]

Cas9n

168

Single plasmid, CBE, ABE

Editing efficiency of most sites>60%

Enhanced resistance to lanthionine antibiotics

[49]

Cas9

BsMN0

Single plasmid, KI

Single amyQ gene integration efficiency 97.5%-98.6%

Multi-gene one-step insertion and selection

[51]

dCas9

1A976

Single plasmid, CRISPRi, CRISPRa

GFP fluorescence decreased by 5.02-8.36 times

Increased expression of recombinant proteins

[52]

I型CRISPRi

168

Single plasmid, CRISPRi

GFP fluorescence decreased by 26.84-34.18 times

The titer of d-pantothenic acid increased to 12.81 g/L

[53]

Cas9

YB886

(168 derivative)

Single plasmid, KO

Enhanced resistance to bacteriophage SPP1

[55]

Cas9

TS01

Single plasmid, KO, KI

KO efficiency >40%; 2 kb KI 5%

Bacteriophage gene editing

[56]

Cas9

ATCC 6051A

Single plasmid,

KO (gene damage)

SrfC 284 bp deletion efficiency 9.1%; spoIIA deletion efficiency 36%; double gene deletion efficiency 33%

Inhibition of foam formation and spore production

[57]

Cre-Cas9

DB428

Dual plasmids, KO, KI

Gene KO and KI efficiency >95%

Insertion of no resistant genes, inactivation of protease genes

[59]

Cas9

dCas9

1A751

Genome integration, KO, KI, CRISPRi

Point mutation 85%-100%; double gene KO 36%; 2.9 kb KI 69%

β-galactosidase activity decreased by 8 times

[60]

dCas9

168

1A751

Genome integration, CRISPRi

FtsZ relative suppression efficiency 25%-100%

Hyaluronic acid production increased by 204%

[60]

Cas9

168

Dual plasmids, KO

500 bp KO >80%

Self-targeted removal of plasmids

[61]

Cas9n

168

Dual plasmids,

point mutation, KO, KI

8 kb KO-82%; 20.5 kb KO-23.6%; 1-2 kb KI >90%

Riboflavin production increased by 59%

[62]

dCas9

168

Genome integration, CRISPRi

GlcNAc production increased 81.7-131.6 g/L

Dynamic regulation of metabolic flux in response to intracellular GlcN6P concentration

[63]

dCas9

BNY

(168 derivative)

Genome integration, CRISPRi

Simultaneously suppressed zwf, pfkA, and glmM intensity 0.01-0.10 times

GlcNAc yield increased by 88.5% with reduced by-products

[63]

dCas9

BY14

(168 derivative)

Genome integration, CRISPRi

Pyk and zwf expression decreased by 13.6 and 12.7 times

Lactose production increased by 59.2%; lactose titer increased by 17.4%

[64]

Cas9n

ATCC6051△5

Single plasmid, CBE

Elimination of resistant genes in plasmids

[65]

System types	Strains	Optimization strategy	Optimization results	References
CRISPRa	SCK6	Inhibited and activated dual regulation	The amylase yield increased by 260 times	[43]
CRISPRi	168	Integration of temperature sensor with CRISPRi system	The production of 2′-fucosyl lactose reached 1 839.7 mg/L	[50]
CRISPRi	168	Reconstruction of type I CRISPRi system to control pdhA expression	The titer of d-pantothenic acid increased to 12.81 g/L	[53]
Cas9	168	Heterologous expression of SeHAS gene, overexpression of pgsA and clsA genes, downregulation of ftsZ gene expression	The hyaluronic acid titer increased by 204%	[54]
CRISPRi	BNY	Inhibition of zwf, pfkA, and glmM genes	The production of N-acetylglucosamine reached 20.5 g/L	[63]
AsCpf1	168	Knockout of eps gene cluster, knockin of tuaD gene at mpr and epr sites	The hyaluronic acid production reached 1.39 g/L	[66]
Cas9	168	Alleviation of transcriptional riboswitch restrictions on riboflavin biosynthesis pathway	The riboflavin production increased by 53%	[67]
Cas9n	168	Regulation of ribB, ribA, and ribH genes	The riboflavin production increased by 59% to reach 1.39 g/L	[68]
dCas12a	S5	Construction of genetic ScrABBLE system	The titer of N-acetylglucosamine reached 183.9 g/L	[69]
Cas9	RLI2019	Knockout of 10 antibiotic resistance genes	The strain was more sensitive to antibiotics	[70-71]
Cas9	RLI2019	Simultaneous integration of eglS, Cel48S, and bglS genes at aprE, epr, and amyE sites	The activities of endoglucanase, exoglucanase, and β-glucosidase were 3.1 times, 6.6 times, and 3.0 times of the starting strain, respectively	[72-74]
Cas9	168	Regulation of key genes in the synthetic pathway trpC2	The lycopene production reached 1.12 mg/L	[75]

System types

Strains

Optimization strategy

Optimization results

References

CRISPRa

SCK6

Inhibited and activated dual regulation

The amylase yield increased by 260 times

[43]

CRISPRi

168