基于CRISPR系统的高通量基因组编辑研究进展

doi:10.12211/2096-8280.2025-073

合成生物学

• 特约评述 •

基于CRISPR系统的高通量基因组编辑研究进展

滕佳尧¹^,²^,³, 任传宏¹^,²^,³, 朱芮莹¹^,²^,³, 鲍泽华¹^,²^,³^,⁴

^1.浙江大学化学工程与生物工程学院，生物质化工教育部重点实验室，浙江杭州 310058
^2.浙江大学杭州国际科创中心，全省功能化学品智造重点实验室，浙江杭州 311215
^3.浙江大学化学工程与生物工程学院，生物工程研究所，浙江杭州 310058
^4.浙江大学化学工程与生物工程学院，浙江省智能生物材料重点实验室，浙江杭州 310058

收稿日期:2025-07-15 修回日期:2025-10-30 出版日期:2025-11-03
通讯作者: 鲍泽华
作者简介:滕佳尧（2002—），男，硕士研究生。研究方向为合成生物学。 E-mail：22428049@zju.edu.cn
任传宏（1993—），男，博士，助理研究员。研究方向为植物天然产物合成调控。E-mail：chuanhongren@163.com
鲍泽华（1988—），男，博士，研究员，博士生导师。研究方向为合成生物学、基因编辑、人工转录因子和定向进化等。 E-mail：zbao@zju.edu.cn
基金资助:
国家重点研发计划(2023YFF1204500);国家自然科学基金(22308316);中央高校基本科研业务费专项资金(226-2025-00043)

Recent advances in CRISPR-based high-throughput genome editing

TENG Jiayao¹^,²^,³, REN Chuanhong¹^,²^,³, ZHU Ruiying¹^,²^,³, BAO Zehua¹^,²^,³^,⁴

^1.Key Laboratory of Biomass Chemical Engineering of Ministry of Education，College of Chemical and Biological Engineering，Zhejiang University，Hangzhou 310058，Zhejiang，China
^2.Zhejiang Key Laboratory of Intelligent Manufacturing for Functional Chemicals，ZJU-Hangzhou Global Scientific and Technological Innovation Center，Zhejiang University，Hangzhou 311215，Zhejiang，China
^3.Institute of Bioengineering，College of Chemical and Biological Engineering，Zhejiang University，Hangzhou 310058，Zhejiang，China
^4.Zhejiang Key Laboratory of Smart Biomaterials，College of Chemical and Biological Engineering，Zhejiang University，Hangzhou 310058，Zhejiang，China

Received:2025-07-15 Revised:2025-10-30 Online:2025-11-03
Contact: BAO Zehua

摘要/Abstract

摘要：

高通量基因组编辑是快速分析大量基因突变功能和进行遗传育种的有效方法。相比于传统随机诱变，基于规律间隔成簇短回文重复序列（CRISPR）系统的基因组编辑具有效率高、可靶向的优点。通过设计靶向目标基因的向导RNA文库可以实现高通量基因组编辑和筛选。近年来，多种CRISPR系统以及CRISPR衍生基因编辑技术的开发进一步丰富了高通量基因组编辑工具箱。本文主要介绍基于CRISPR系统的高通量基因组编辑方法，包括CRISPR辅助的同源定向修复、碱基编辑系统、引导编辑系统等，并介绍了这些方法在不同领域的应用，如工业微生物育种、人类功能基因组学和作物改良。最后，对相关方法存在的物种适用性有限、突变多样性低、编辑范围窄、多基因编辑困难等问题以及潜在的解决方法进行讨论和展望。

关键词: CRISPR, 基因组编辑, 高通量, 同源定向修复, 碱基编辑, 引导编辑

Abstract:

High-throughput genome editing is an effective approach to rapidly analyzing the function of massive genetic mutations and to performing genetic breeding. Compared with random mutagenesis, the Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing is more efficient and programmable. High-throughput genome editing and screening is enabled by the design of guide RNA libraries targeting specific genes. In recent years, the high-throughput genome editing toolbox is enriched by various CRISPR systems and CRISPR-derived technologies. Here we review major CRISPR-based high-throughput genome editing methods, including CRISPR-assisted homology directed repair, base editing systems, and prime editing systems, and discuss their applications in different fields, including industrial microbial strain breeding, functional human genomics research and crop improvement. These methods were applied in enhancing the tolerance and production capacity of microorganisms in industrial microbial strain breeding, analyzing the functions of disease-associated single nucleotide variants (SNVs) in functional human genomics research, and enhancing the herbicide resistance of plants in crop improvement. To conclude, we discuss the limitations of these methods, including the limited species applicability, the low mutation diversity, the narrow editing window, and the difficulty in multiplex genome editing. We provide prospects to address these limitations, including, firstly, expanding the applicable species from model organisms such as Escherichia coli, Saccharomyces cerevisiae to other important industrial microorganisms such as Actinomycetes and Pseudomonas aeruginosa by using related CRISPR systems; secondly, increasing mutation diversity by developing more advanced editors, particularly for base editors; thirdly, broadening the targeting region of genome editors by using PAM-relaxed or computationally designed Cas variants, as well as novel base editor and prime editor architectures; fourthly, scaling up multiplex genome editing for more targeted sites. With the development of artificial intelligence and automation platforms, as well as the continued rapid advancement of CRISPR and its derivative technologies, we expect that more advanced high-throughput genome editing technologies will emerge.

Key words: CRISPR, genome editing, high-throughput, homology directed repair, base editing, prime editing

中图分类号:

Q812

滕佳尧, 任传宏, 朱芮莹, 鲍泽华. 基于CRISPR系统的高通量基因组编辑研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2025-073.

TENG Jiayao, REN Chuanhong, ZHU Ruiying, BAO Zehua. Recent advances in CRISPR-based high-throughput genome editing[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2025-073.

图/表 8

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编辑方法	方法名称	物种	Cas蛋白	PAM	效应蛋白	参考文献
CRISPR-HDR	Saturation editing	哺乳动物细胞	SpCas9	NGG	/	[38]
	CasPER	酿酒酵母	SpCas9	NGG	/	[43]
	CREATE	大肠杆菌	SpCas9	NGG	/	[44, 45]
	CHAnGE	酿酒酵母	SpCas9	NGG	/	[46]
	MAGESTIC	酿酒酵母	SpCas9	NGG	/	[47]
	CRISPEY	酿酒酵母	SpCas9	NGG	/	[49]
	CHASE	酿酒酵母	SpiG（SpCas9突变体）	NGN	/	[50]
	CRAIDE	酿酒酵母	SpCas9	NGG	/	[52]
Base editing	TAM	哺乳动物细胞	dSpCas9	NGG	胞苷脱氨酶AID-P182X（AIDx）	[63]
	CRISPR-X	哺乳动物细胞	dSpCas9	NGG	胞苷脱氨酶AID/AID*∆	[64]
	BARBEKO	哺乳动物细胞	nSpCas9	NGG	胞苷脱氨酶Anc689 APOBEC	[65]
	HACE	哺乳动物细胞	nSpCas9	NGG	胞苷脱氨酶AID*∆ 解旋酶PcrA M6	[66]
	MACBETH	谷氨酸棒状杆菌	nSpCas9	NGG	胞苷脱氨酶AID-P47S（AID^TS）	[68]
	CoMuTER	酿酒酵母	Cas3	AAG	胞苷脱氨酶rAPOBEC1	[69]
	ScBE3	大肠杆菌	nScCas9	NNG	胞苷脱氨酶rAPOBEC1	[70]
	STEME STEME-NG	水稻	nSpCas9 nCas9-NG	NGG NG	胞苷脱氨酶APOBEC3 腺苷脱氨酶ecTadA-ecTadA7.10	[71]
	MoBE	水稻	nSpCas9	NGG	胞苷脱氨酶CDA1 腺苷脱氨酶TadA9	[72]
Prime editing	PLSM	水稻	nSpCas9	NGG	逆转录酶MMLV-RT（基于pPE2）	[73]
	SPE	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE2）	[74]
	PRIME	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE3）	[75]
	PEER-seq	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[76]
	MOSAIC	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE2）	[77]
	高通量基因敲除筛选的PE平台	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[80]
	PRESENT	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[81]

编辑方法	方法名称	物种	Cas蛋白	PAM	效应蛋白	参考文献
CRISPR-HDR	Saturation editing	哺乳动物细胞	SpCas9	NGG	/	[38]
	CasPER	酿酒酵母	SpCas9	NGG	/	[43]
	CREATE	大肠杆菌	SpCas9	NGG	/	[44, 45]
	CHAnGE	酿酒酵母	SpCas9	NGG	/	[46]
	MAGESTIC	酿酒酵母	SpCas9	NGG	/	[47]
	CRISPEY	酿酒酵母	SpCas9	NGG	/	[49]
	CHASE	酿酒酵母	SpiG（SpCas9突变体）	NGN	/	[50]
	CRAIDE	酿酒酵母	SpCas9	NGG	/	[52]
Base editing	TAM	哺乳动物细胞	dSpCas9	NGG	胞苷脱氨酶AID-P182X（AIDx）	[63]
	CRISPR-X	哺乳动物细胞	dSpCas9	NGG	胞苷脱氨酶AID/AID*∆	[64]
	BARBEKO	哺乳动物细胞	nSpCas9	NGG	胞苷脱氨酶Anc689 APOBEC	[65]
	HACE	哺乳动物细胞	nSpCas9	NGG	胞苷脱氨酶AID*∆ 解旋酶PcrA M6	[66]
	MACBETH	谷氨酸棒状杆菌	nSpCas9	NGG	胞苷脱氨酶AID-P47S（AID^TS）	[68]
	CoMuTER	酿酒酵母	Cas3	AAG	胞苷脱氨酶rAPOBEC1	[69]
	ScBE3	大肠杆菌	nScCas9	NNG	胞苷脱氨酶rAPOBEC1	[70]
	STEME STEME-NG	水稻	nSpCas9 nCas9-NG	NGG NG	胞苷脱氨酶APOBEC3 腺苷脱氨酶ecTadA-ecTadA7.10	[71]
	MoBE	水稻	nSpCas9	NGG	胞苷脱氨酶CDA1 腺苷脱氨酶TadA9	[72]
Prime editing	PLSM	水稻	nSpCas9	NGG	逆转录酶MMLV-RT（基于pPE2）	[73]
	SPE	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE2）	[74]
	PRIME	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE3）	[75]
	PEER-seq	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[76]
	MOSAIC	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PE2）	[77]
	高通量基因敲除筛选的PE平台	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[80]
	PRESENT	哺乳动物细胞	nSpCas9	NGG	逆转录酶MMLV-RT（基于PEmax）	[81]

基于CRISPR系统的高通量基因组编辑研究进展

Recent advances in CRISPR-based high-throughput genome editing

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