CRISPR/Cas系统的挖掘、改造与功能拓展

柳柯; 林桂虹; 刘坤; 周伟; 王风清; 魏东芝

doi:10.12211/2096-8280.2021-022

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CRISPR/Cas系统的挖掘、改造与功能拓展

特约评述 | 更新时间：2025-03-20

- CRISPR/Cas系统的挖掘、改造与功能拓展
- Mining, engineering and functional expansion of CRISPR/Cas systems
- 合成生物学 2023年4卷第1期页码：47-66
- 作者机构：
  
  华东理工大学生物反应器工程国家重点实验室，鲁华生物技术研究所，上海 200237
- 作者简介：
  
  [ "柳柯（1995—），男，博士研究生。研究方向为基因组编辑技术的开发与应用等。E-mail：y12190016@mail.ecust.edu.cn" ]
  [ "王风清（1977—），男，副教授，博士生导师。研究方向为利用代谢工程和合成生物学的原理和方法，致力于微生物细胞工厂的研究和开发等。E-mail：fqwang@ecust.edu.cn" ]
  [ "魏东芝（1963—），男，二级教授，博士生导师。研究方向为生物元器件的发现、改造与应用研究，致力于发现和改进具有工业应用价值的微生物和生物催化剂，开拓生物转化新反应等。E-mail：dzhwei@ecust.edu.cn" ]
- 基金信息：
  
  国家自然科学基金(21978084)
- DOI：10.12211/2096-8280.2021-022
  中图分类号： Q816
- 收稿：2021-02-08，
  
  修回：2021-04-28，
  
  纸质出版：2023-02-28
- 稿件说明：
移动端阅览
柳柯，林桂虹，刘坤，周伟，王风清，魏东芝. CRISPR/Cas系统的挖掘、改造与功能拓展［J］. 合成生物学， 2023， 4（1）： 47-66

LIU Ke， LIN Guihong， LIU Kun， ZHOU Wei， WANG Fengqing， WEI Dongzhi. Mining， engineering and functional expansion of CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 47-66
柳柯，林桂虹，刘坤，周伟，王风清，魏东芝. CRISPR/Cas系统的挖掘、改造与功能拓展［J］. 合成生物学， 2023， 4（1）： 47-66 DOI： 10.12211/2096-8280.2021-022.

LIU Ke， LIN Guihong， LIU Kun， ZHOU Wei， WANG Fengqing， WEI Dongzhi. Mining， engineering and functional expansion of CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 47-66 DOI： 10.12211/2096-8280.2021-022.

摘要

规律成簇间隔短回文重复序列及其相关蛋白（CRISPR/Cas）是一种微生物获得性免疫系统，自从证实其可用于基因编辑之后，迅速增强了我们编辑、操纵、注释、检测甚至成像生物体DNA和RNA的能力，为基础生命科学、医学和生物工程等领域的创新发展注入了强劲动力，快速推动了合成生物学等学科的兴盛发展。然而，CRISPR/Cas系统也有一些固有的问题，例如脱靶效应、原间隔序列邻近基序（PAM）对靶目标的约束性以及基因编辑活性的可控性等，严重制约了该系统在基因精准可控编辑等方面的长足发展，阻碍了其新功能和新应用的拓展。为了突破这些限制，“蛋白质工程修饰Cas蛋白”与“基于生物信息学的新型CRISPR/Cas系统的挖掘”就成为完善发展CRISPR/Cas系统以及扩充CRISPR工具箱的两种重要策略。本文主要针对当前应用最为广泛的Ⅱ类CRISPR/Cas系统，重点介绍了CRISPR/Cas9、CRISPR/Cas12a和CRISPR/Cas13a这三种代表性系统的基本结构和作用机制，及其在结构改造和功能拓展等方面的新进展，同时也对一些新近发掘的具有重要特色和潜在应用价值的CRISPR/Cas系统进行了综述，例如CRISPR/CasФ 和 CRISPR/Cas12k。这些改造和发掘工作显著改善了CRISPR/Cas系统的固有问题，有力地拓展了其功能和适用性，势必会进一步快速推动CRISPR/Cas系统在诸多领域的创新发展。

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) were derived from an acquired immune system in microbes. Since their functions on gene editing have been reported

they have been rapidly used to enhance our ability to edit

regulate

annotate

detect

and image DNA and RNA fragments of various organisms

which consequently have faciliated fundamental research in life science

medicine

bioengineering and so on

and drived the development of synthetic biology and other disciplines. However

CRISPR/Cas systems also have some inherent drawbacks

such as off-target effect

constraint of protospacer-adjacent motif (PAM) on the target

and controllability of the gene editing activity

which substantially compromise their applications in highly precise and controllable gene editing. In order to overcome these challenges

two important strategies have been employed to develop enhanced CRISPR/Cas systems and expand the CRISPR toolbox

including modifying the Cas proteins by protein engineering and mining novel CRISPR/Cas systems with bioinformatics. In the review

focusing on the most widely used the type II CRISPR/Cas systems

we mainly introduce the basic structures and functions of three representative systems

including CRISPR/Cas9

CRISPR/Cas12a and CRISPR/Cas13a

as well as recent progress in their structural modifications and functional expansion. Thereinto

engineering strategies for CRISPR/Cas systems have been systematically commented

which include modification methods for Cas proteins and the way to expand the function of CRISPR/Cas systems by coupling specfic proteins with Cas. In addition

we also review some novel CRISPR/Cas systems with important characteristics and potential applications that have been discovered in recently years

such as CRISPR/CasФ and CRISPR/Cas12k. These engineering modifications and mining work have greatly addressed the inherent problems of the CRISPR/Cas systems

and effectively expanded their functions and applicability

which will further promote the applications of the CRISPR/Cas systems in many fields.

关键词

Keywords

references

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