合成生物学 ›› 2023, Vol. 4 ›› Issue (4): 720-737.DOI: 10.12211/2096-8280.2022-053

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碱基编辑技术及其在微生物合成生物学中的应用

王雁南, 孙宇辉   

  1. 武汉大学药学院,组合生物合成与新药发现教育部重点实验室,湖北 武汉 430071
  • 收稿日期:2022-09-28 修回日期:2022-11-13 出版日期:2023-08-31 发布日期:2023-09-14
  • 通讯作者: 孙宇辉
  • 作者简介:王雁南(1994—),男,博士。研究方向为基因编辑工具开发和优化。E-mail:ynw@whu.edu.cn
    孙宇辉(1967—),男,博士,教授,博士生导师。研究方向为微生物药物合成生物学及其编辑工具开发和优化。E-mail:yhsun@whu.edu.cn
  • 基金资助:
    国家重点研发计划合成生物学重点专项(2018YFA0903200)

Base editing technology and its application in microbial synthetic biology

Yannan WANG, Yuhui SUN   

  1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education),School of Pharmaceutical Sciences,Wuhan University,Wuhan 430071,Hubei,China
  • Received:2022-09-28 Revised:2022-11-13 Online:2023-08-31 Published:2023-09-14
  • Contact: Yuhui SUN

摘要:

CRISPR/Cas系统的发现与发展对生命科学领域产生了革命性的影响,借助CRISPR/Cas系统研发出的一系列工具为相关领域的研究带来了极大的便利。碱基编辑器便是其中一类基于CRISPR/Cas系统开发的可实现碱基转换和颠换的基因编辑工具,其通过将胞苷或腺苷脱氨酶以及其他功能元件与失去双链切割活性的Cas蛋白相融合,由sgRNA引导,实现对基因组上目标位置胞嘧啶或腺嘌呤的碱基转换。碱基编辑器一经开发便在生物学、医学等领域展现出巨大的应用潜力,虽然经过不断优化,但目前在使用时仍然存在着许多制约因素。本文简述了几种主要碱基编辑器的发展,并介绍了碱基编辑器存在的靶向范围受限和脱靶编辑的问题以及现有的优化措施。同时列举了我国部分科研工作者将碱基编辑技术运用于微生物合成生物学领域的进展,并展望了碱基编辑技术的发展及其在微生物合成生物学领域的应用前景。

关键词: 碱基编辑器, 特异性优化, 靶向范围拓展, 微生物, 合成生物学

Abstract:

The discovery and development of the CRISPR/Cas system have a revolutionary influence on life sciences. A series of tools derived from the CRISPR/Cas system have brought great convenience to research in the field of life sciences. The base editors developed based on the CRISPR/Cas system are gene editing tools that can achieve base conversions and transversion on target. The base editors are constructed by fusing cytosine or adenosine deaminase, and other functional elements to Cas proteins with abolished double strand DNA cleavage activity to convert cytidine or adenine into other bases at genome on-target sites guided by sgRNAs. Base editors have shown great potential in biology, medicine and related fields. Although they have already been continuously optimized, there are still problems affecting further application of base editors. In this review, we briefly describe the development of DNA base editors. Furthermore, we introduce in detail the problems of the limited editing range of base editors as well as the corresponding optimization strategies by increasing the target sites recognized by the locator moiety and expanding or narrowing the editing window of the effector moiety. At the same time, we introduce several off-target editing detection methods specially developed for base editing. Based on usual and developed detection methods, multiple and frequent off-target editing caused by base editors were found at both DNA and RNA levels. We also introduced various effective optimization strategies to improve the editing specificity of the base editors in every respect. Most of these strategies are based on protein modification, but also on optimization of sgRNA and spatio-temporal regulation of base editing systems. These measures greatly enrich the application scenarios of the base editors. Then, we discuss the progress on applying base editors to the field of microbial synthetic biology, including revealing the metabolic pathway and synthesis mechanism of natural products as well as improving the production of target compounds in multiple species. Finally, we envisage the promising development of base editing in synthetic biology in the future. {L-End}

Key words: base editors, improvement of specificity, expansion of target range, microorganism, synthetic biology

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