Optimization and development of CRISPR/Cas9 systems for genome editing

TENG Xiaolong; SHI Shuobo

doi:10.12211/2096-8280.2022-047

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Optimization and development of CRISPR/Cas9 systems for genome editing

Invited Review | 更新时间：2025-03-20

- Optimization and development of CRISPR/Cas9 systems for genome editing
  封面论文
- Synthetic Biology Journal Vol. 4, Issue 1, Pages: 67-85(2023)
- 作者机构：
  
  北京化工大学，北京软物质科学与工程高精尖创新中心，北京 100029
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2022-047
  CLC： Q819
- Received：01 September 2022，
  
  Revised：2022-10-28，
  
  Published：28 February 2023
- 稿件说明：
移动端阅览
滕小龙，史硕博. CRISPR/Cas9系统在基因组编辑中的优化与发展［J］. 合成生物学， 2023， 4（1）： 67-85

TENG Xiaolong， SHI Shuobo. Optimization and development of CRISPR/Cas9 systems for genome editing［J］. Synthetic Biology Journal， 2023， 4（1）： 67-85
滕小龙，史硕博. CRISPR/Cas9系统在基因组编辑中的优化与发展［J］. 合成生物学， 2023， 4（1）： 67-85 DOI： 10.12211/2096-8280.2022-047.

TENG Xiaolong， SHI Shuobo. Optimization and development of CRISPR/Cas9 systems for genome editing［J］. Synthetic Biology Journal， 2023， 4（1）： 67-85 DOI： 10.12211/2096-8280.2022-047.

摘要

CRISPR/Cas9是近年来发展起来的新兴技术，其在多种生物和组织的基因组上具有快速、高效、精准的基因编辑与调控能力，这使得该技术在基础科学和合成生物学等应用科学领域均得到了极大的发展与应用。本文首先对CRISPR的历史沿革、分类及CRISPR/Cas9技术的作用机制进行简述，并结合其原理和在基因组工程中面临的脱靶率高、PAM依赖性强等限制因素总结了近年来针对Cas9蛋白和向导RNA（gRNA）进行的一系列优化与改造。接下来详细叙述了CRISPR/Cas9系统结合效应蛋白实现的多种功能，包括基因表达调控、表观基因组编辑、单碱基编辑等。基于gRNA多表达策略和Cas9多路复用策略，本文还对CRISPR/Cas9技术主要的多重应用成果进行了梳理汇总。最后探讨了CRISPR/Cas9作为高精度基因组编辑工具使用的应用前景，以及作为疗法使用时的安全性和风险控制问题。

Abstract

As an emerging technology developed within recent years

CRISPR/Cas9 exhibits fast

efficient

and precise gene editing and regulation capabilities in various organisms and tissues

and these advantages make it widely used in research with fundamental sciences and applied technologies as well such as synthetic biology. This review first briefly introduces the discovery history

classification

and mechanism of CRISPR/Cas9. The system of CRISPR/Cas9 usually contains a single guide RNA (gRNA) molecule for targeting a specific sequence

and a Cas9 endonuclease for catalyzing a double-strand break (DSB) in the sequence (target DNA strands). The recognition and cleavage of target DNA strictly require the presence of a protospacer adjacent motif (PAM) in the target sequence. The DSB(s) can be repaired by various DNA repair mechanisms

which allow various gene editing such as gene integration

gene replacement

and gene knockout. Due to limitations of CRISPR/Cas9

such as PAM dependence and high off-target rate

researchers have developed various fused or engineered Cas9 proteins and gRNAs that play significant roles in fulfilling various purposes. These Cas9 variants are modified for improving the performance of PAM

in particular its specificity and fidelity. Moreover

the DSBs generated by Cas9 are considered toxic to the cells

and the use of Cas9 nickase (nCas9) or catalytically deficient Cas9 (dCas9) in CRISPR has also been developed without generating DSBs. Meanwhile

different effector proteins can be fused with Cas9/dCas9/nCas9 to bring about new functions and applications in gene expression regulation

epigenome editing

and single base editing. Moreover

we introduce the current studies and applications of multiple gRNA expression strategies based on the multiplex advantages of the CRISPR/Cas9 system. In general

CRISPR/Cas9 systems have gradually become standardized and revolutionized genome editing systems for almost all possible genetic manipulations. Finally

we highlight perspectives on several applications of the versatile CRISPR/Cas9 toolbox as a genome editing tool

and discuss the safety and risk control issues when it is used in gene therapy.

关键词

Keywords

references

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