CRISPR/Cas9基因编辑技术在微生物细胞中的应用研究进展

Application progress of genome editing technology CRISPR/Cas9 in microbial cells

CRISPR/Cas系统(clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)是原核生物中广泛存在的、用于抵御外源遗传物质入侵的获得性免疫防御系统.由II型CRISPR/Cas系统改造而来的CRISPR/Cas9技术,是继锌指核酸酶和转录激活样效应因子核酸酶后的第三代基因编辑技术.基于近年文献综述CRISPR/Cas9系统的发现、免疫防御过程和作用机制,并总结其在微生物细胞中的应用研究进展.CRISPR/Cas9系统由tracrRNA、crRNA和Cas9蛋白组成的复合物识别PAM序列并行使切割功能,不同微生物来源的CRISPR/Cas9系统识别的PAM序列存在差异;CRISPR/Cas9技术对微生物尤其是传统遗传操作较难微生物的基因组编辑,例如碱基或大片段的插入、缺失或突变等更为简单、高效和易操作;CRISPR/Cas9以及CRISPRa和CRISPRi技术,在原核和真核微生物基因表达调控、代谢工程等领域具有巨大的应用潜力,如医药、食品以及工业等相关重要产品的获得.未来,结合微生物组学大数据等,需继续挖掘不同微生物来源的CRISPR/Cas系统或其他新型基因编辑工具,并对已有CRISPR/Cas9系统继续优化升级,降低脱靶率、提高编辑效率并扩大CRISPR/Cas9的应用范围.(图1表1参72)

The clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins(CRISPR/Cas)system is an acquired immune defense system that is widely distributed in prokaryotic organisms and defends against the invasion of exogenous genetic materials.Modified from the type II CRISPR/Cas system,CRISPR/Cas9 is the third generation of genome site-specific editing technology,after zinc finger nucleases and transcription activator-like effector nucleases.Based on recent research,this paper reviews the discovery,immune defense,and mechanisms of CRISPR/Cas9,and summarizes its applications in microbial cells.The CRISPR/Cas9 system recognizes PAM sequences and performs cleavage functions by a complex comprising tracrRNA,crRNA,and Cas9,and the PAM sequences are different in different microorganisms.CRISPR/Cas9 technology is simple,efficient,and easy for genome editing of microorganisms,such as base or large fragment insertion,deletion,or mutation,especially those that are difficult to manipulate using the traditional genetic operation method.CRISPR/Cas9,CRISPRa,and CRISPRi have great application potential in prokaryotes and eukaryotes for the regulation of gene expression,metabolic engineering,and in fields,such as the acquisition of important products related to medicine,food,and industry.In the future,more CRISPR/Cas systems or new genome editing tools should be continuously explored with the help of big data on microbiomes.The existing CRISPR/Cas9 system should be continuously optimized and upgraded to reduce off-target effects,improve editing efficiency,and expand its application scope.