Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems

LIANG Liya; LIU Rongming

doi:10.12211/2096-8280.2022-040

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Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems

Invited Review | 更新时间：2025-03-20

- Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems
- Synthetic Biology Journal Vol. 4, Issue 1, Pages: 86-101(2023)
- 作者机构：
  
  大连理工大学生物工程学院，辽宁大连 116024
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2022-040
  CLC： Q812
- Received：18 July 2022，
  
  Revised：2022-09-28，
  
  Published：28 February 2023
- 稿件说明：
移动端阅览
梁丽亚，刘嵘明. 靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造［J］. 合成生物学， 2023， 4（1）： 86-101

LIANG Liya， LIU Rongming. Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 86-101
梁丽亚，刘嵘明. 靶向DNA的Ⅱ类CRISPR/Cas系统的蛋白工程化改造［J］. 合成生物学， 2023， 4（1）： 86-101 DOI： 10.12211/2096-8280.2022-040.

LIANG Liya， LIU Rongming. Protein engineering of DNA targeting type Ⅱ CRISPR/Cas systems［J］. Synthetic Biology Journal， 2023， 4（1）： 86-101 DOI： 10.12211/2096-8280.2022-040.

摘要

Clustered regularly interspaced short palindromic repeats （CRISPR）/CRISPR-associated （Cas） protein是细菌或古细菌特有的一种获得性免疫系统，自从研究人员将其改造为基因编辑工具之后，CRISPR/Cas系统高效的基因编辑能力对生命科学、生物工程、生物医药、食品及农业科学等多个领域引发了革命性的影响。然而，研究者们发现CRISPR/Cas系统存在脱靶效应、PAM位点识别范围有限等问题，限制了CRISPR/Cas的应用。为了解决这些问题，针对Cas蛋白进行工程化改造已经成为了优化及开发CRISPR/Cas系统的重要策略。本文以Ⅱ类CRISPR/Cas系统中靶向DNA的CRISPR/Cas9以及CRISPR/Cas12a为例，聚焦近年来针对Cas9和Cas12a蛋白的优化改造方法及相关进展进行系统阐述总结，包括Cas蛋白的工程化改造提升基因编辑特异性及改变PAM识别能力，以CRISPR/Cas系统作为基因定位工具开发新功能，结合外源蛋白调控CRISPR/Cas系统功能。这些工作开发了系列高特异性、高精度的CRISPR/Cas系统，极大地扩展了CRISPR/Cas系统的功能及适用范围，为CRISPR/Cas系统的多领域应用做出了重要贡献。

Abstract

With different types of nucleases

genome editing technologies have opened up the possibility for targeting and modifying specific gene sequences

which show potential applications in basic and applied aspects of biotechnology research. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are artificial proteins generated by fusing a specific DNA-binding domain with a restriction enzyme FokI DNA-cleavage domain

which arise from their ability to customize the DNA-binding domain for recognition of targeting sequences and cleaving them by the FokI domain. However

the design and construction of such a system are time consuming

laborious and costly. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) protein is a unique acquired immune system of bacteria or archaea. Since researchers constructed the CRISPR/Cas system for gene editing

its high efficiency has revolutionized a variety of fields such as life sciences

bioengineering

biomedicine

food

and agricultural sciences. However

the CRISPR/Cas system still has some challenges

such as off-target effect and limited PAM site recognition range

which limit its further applications. In order to solve these problems

molecular engineering of Cas proteins has become an important strategy for developing and optimizing CRISPR/Cas systems. In this study

with CRISPR/Cas9 and CRISPR/Cas12a selected as representative examples for DNA-targeting Class II CRISPR/Cas systems

we focus on the optimization and modification methods

and progress of Cas9 and Cas12a proteins achieved within recent years

such as Cas protein engineering for improved on-target specificity and expanded PAM scopes

developing new functions using CRISPR/Cas systems as gene targeting tools

and introducing exogenous protein domains to regulate CRISPR/Cas functions. These studies have generated a series of high-specificity and high-precision CRISPR/Cas systems

which have greatly expanded their functions and scopes

and made important contributions to the wide-range applications of CRISPR/Cas systems.

关键词

Keywords

references

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Biosafety strategies for engineered bacteria

Related Author

Rongming LIU

Liya LIANG

LI Yang

SHEN Xiaolin

SUN Xinxiao

YUAN Qipeng

YAN Yajun

WANG Jia

Related Institution

College of Engineering， The University of Georgia

State Key Laboratory of Chemical Resource Engineering，Beijing University of Chemical Technology

College of Engineering， The University of Georgia

Key Laboratory of Systems Bioengineering （Ministry of Education）， Frontier Science Center for Synthetic Biology， Tianjin University

School of Chemical Engineering and Technology， Tianjin University

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