Application and optimization of CRISPR editing technology in Streptomyces

doi:10.11949/0438-1157.20240895

Abstract

Abstract:

Streptomyces chassis development is an effective strategy for the high-efficient synthesis of microbial source nature product, and highly efficient gene editing tools provide a powerful tool for the construction of Streptomyces cell factories. CRISPR (clustered regularly interspaced short palindromic repeats) technology is accurate, universal and easy to operate, and has been widely used in microorganisms. However, off-target effects, cytotoxicity and low editing efficiency limit application of CRISPR genome editing in Streptomyces. This article summarizes the application and optimization strategies of CRISPR-related technologies in Streptomyces, and looks forward to the application prospects of CRISPR systems in Streptomyces cell factories, providing a reference for the development of efficient gene editing tools suitable for Streptomyces.

Key words: CRISPR, Streptomyces, gene editing, biological techniques, optimization

摘要：

链霉菌底盘细胞的开发是微生物源药物高效合成的有效策略，高效的基因编辑工具为构建链霉菌细胞工厂提供了有力的技术支撑。CRISPR（clustered regularly interspaced short palindromic repeats）技术具有精准性、普适性和易操作性，在微生物中得到了广泛的应用。然而脱靶效应、Cas蛋白毒性、编辑效率低下等原因，导致CRISPR在链霉菌中的应用受到一定的限制。对CRISPR相关技术在链霉菌中的应用及优化策略进行了总结和归纳，并对CRISPR系统在链霉菌细胞工厂中的应用前景进行了展望，为开发适用于链霉菌的高效基因编辑工具提供了参考。

关键词: CRISPR, 链霉菌, 基因编辑, 生物技术, 优化策略

CLC Number:

Q 812

Xinying LI, Chang SU, Chao GUO, Jian PANG, Chao WANG, Chun LI. Application and optimization of CRISPR editing technology in Streptomyces[J]. CIESC Journal, 2025, 76(3): 922-932.

李新颖, 苏畅, 郭超, 庞建, 王超, 李春. CRISPR技术在链霉菌细胞工厂中的应用和优化[J]. 化工学报, 2025, 76(3): 922-932.

Figures/Tables 4

References 104

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优化策略	改进原理	改进说明	宿主	编辑效率	文献
Cas蛋白改造	减小内切酶活性	NickCas9（D10A）	S. rapamycinicus	27.2%~30%	[74]
Cas蛋白改造	拓宽PAM位点识别范围	FnCas12a3（EP16）	S. roseosporus	50.0%±12.5%	[35]
Cas蛋白筛选	降低Cas蛋白毒性	FnCpf1	S. coelicolor M145	87%	[12]
		SaCas9	Streptomyces sp. NRRL S-244	100%	[67]
		Sth1Cas9	S. albus J1074	100%	[68]
		AsCas12j-2	S. coelicolor A3(2)	100%	[72]
		AsCas12f1	S. coelicolor M145	100%	[73]
		AsCas12f1	S. hygroscopicus SIPI-054	67%~83%	[73]
		内源：SviCas3	S. virginiae IBL14	—	[77]
		内源：Type Ⅰ-E CRISPR/Cas系统	S. avermitilis	—	[78]
表达水平调控	调控Cas蛋白的表达水平	CUmate诱导系统	S. coelicolor M145	65%	[79]
		核糖体开关：茶碱诱导	S. coelicolor M145	87.5%	[83]
		纤维素二糖诱导系统	S. coelicolor M1146	—	[81]
			S. lividans TK24	—
			S. albidoflavus J1074	—
		开关诱导型启动子tipA	S. coelicolor M145	80%	[83]
		蓝光激活重构	S. coelicolor M145	80%	[83]
		抗CRISPR抑制剂AcrIIA4	S. coelicolor M145	65%~90%	[85]
修复效率	提高同源重组效率	过表达AtpD	S. coelicolor M145	80%	[83]
	提高同源重组效率	过表达RecA	Nonomuraea gerenzanensis	100%	[87]
	NHEJ途径	Msm-LK、Sda-LK和Ppu-LK	S. coelicolor M145	20%、43.3%、56.7%	[12]
转化效率	系统构建方法优化	诱饵DNA、Cas9和sgRNA表达质粒	S. ambofaciens	15%~20%	[89]
反向筛选	显色反应	GusA	S. rimosus	100%	[90]
	显色反应	IdgS	Verrucosispora sp. MS100137	100%	[91]
	致死效应	CodA(sm)	S. coelicolor M145	95%~99%	[11]

优化策略	改进原理	改进说明	宿主	编辑效率	文献
Cas蛋白改造	减小内切酶活性	NickCas9（D10A）	S. rapamycinicus	27.2%~30%	[74]
Cas蛋白改造	拓宽PAM位点识别范围	FnCas12a3（EP16）	S. roseosporus	50.0%±12.5%	[35]
Cas蛋白筛选	降低Cas蛋白毒性	FnCpf1	S. coelicolor M145	87%	[12]
		SaCas9	Streptomyces sp. NRRL S-244	100%	[67]
		Sth1Cas9	S. albus J1074	100%	[68]
		AsCas12j-2	S. coelicolor A3(2)	100%	[72]
		AsCas12f1	S. coelicolor M145	100%	[73]
		AsCas12f1	S. hygroscopicus SIPI-054	67%~83%	[73]
		内源：SviCas3	S. virginiae IBL14	—	[77]
		内源：Type Ⅰ-E CRISPR/Cas系统	S. avermitilis	—	[78]
表达水平调控	调控Cas蛋白的表达水平	CUmate诱导系统	S. coelicolor M145	65%	[79]
		核糖体开关：茶碱诱导	S. coelicolor M145	87.5%	[83]
		纤维素二糖诱导系统	S. coelicolor M1146	—	[81]
			S. lividans TK24	—
			S. albidoflavus J1074	—
		开关诱导型启动子tipA	S. coelicolor M145	80%	[83]
		蓝光激活重构	S. coelicolor M145	80%	[83]
		抗CRISPR抑制剂AcrIIA4	S. coelicolor M145	65%~90%	[85]
修复效率	提高同源重组效率	过表达AtpD	S. coelicolor M145	80%	[83]
	提高同源重组效率	过表达RecA	Nonomuraea gerenzanensis	100%	[87]
	NHEJ途径	Msm-LK、Sda-LK和Ppu-LK	S. coelicolor M145	20%、43.3%、56.7%	[12]
转化效率	系统构建方法优化	诱饵DNA、Cas9和sgRNA表达质粒	S. ambofaciens	15%~20%	[89]
反向筛选	显色反应	GusA	S. rimosus	100%	[90]
	显色反应	IdgS	Verrucosispora sp. MS100137	100%	[91]
	致死效应	CodA(sm)	S. coelicolor M145	95%~99%	[11]

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