STEME系统：一种助力体内定向进化的新工具

doi:10.16288/j.yczz.20-041

遗传 ›› 2020, Vol. 42 ›› Issue (3): 231-235.doi: 10.16288/j.yczz.20-041

• 前沿聚焦 • 下一篇

STEME系统：一种助力体内定向进化的新工具

胡风越, 王克剑()

中国水稻研究所,水稻生物学国家重点实验室,杭州 310006

收稿日期:2020-02-17 修回日期:2020-02-26 出版日期:2020-03-20 发布日期:2020-02-26
通讯作者: 王克剑 E-mail:wangkejian@caas.cn
作者简介:胡风越,硕士研究生,专业方向：作物种质资源学。E-mail: hufy_caas@163.com
基金资助:
国家转基因专项资助编号(2019ZX08010-003)

The STEME system: a novel tool for directed evolution in vivo

Fengyue Hu, Kejian Wang()

State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China

Received:2020-02-17 Revised:2020-02-26 Online:2020-03-20 Published:2020-02-26
Contact: Wang Kejian E-mail:wangkejian@caas.cn
Supported by:
Supported by the National Transgenic Science and Technology Program No(2019ZX08010-003)

摘要/Abstract

摘要：

通过定向进化(directed evolution)可以快速进行蛋白工程改良及重要基因功能研究,以获得新型农艺性状突变体。近期,中国科学院遗传与发育生物学研究所高彩霞团队和李家洋团队合作构建了新型的饱和靶向内源诱变编辑器(saturated targeted endogenous mutagenesis editors, STEMEs),并在植物中实现了基因的定向进化和功能筛选。该系统融合了现有的2种单碱基编辑技术,成功实现在植物体内同时诱导C:G>T:A、A:T>G:C双碱基编辑,通过靶向OsACC羧基转移酶结构域编码序列定向进化出水稻除草剂抗性植株。这种在体内进行基因定向进化的新方法,对于今后农作物重要农艺性状的筛选和功能基因研究具有重要作用。本文对STEME系统的组成、编辑效率和应用原理进行介绍,并与已有的定向进化方法进行比较,为加速作物种质资源创新研究提供参考。

关键词: STEME, 双碱基编辑, 定向进化, 种质创新

Abstract:

Directed evolution can be rapidly applied for engineering proteins, studying gene functions, and obtaining mutants with important agronomic traits. Recently, Caixia Gao and Jiayang Li’s team from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, worked together to engineer novel saturated targeted endogenous mutagenesis editors (STEMEs), realizing in vivo directed evolution and function selection in plants. This system integrated the existing two single-base editing techniques, successfully induced C:G>T:A and A:T>G:C double-base editing in plants, and artificially evolved into herbicide-resistant rice through targeting the OsACC carboxyltransferase domain coding sequence. This new method of gene directed evolution in vivo displays great application potential in important agronomic trait screening and plant functional gene researches. Here we introduce the composition, editing efficiency, and application principle of the STEME system, and compare it with the existing directed evolution methods, so as to provide a reference for accelerating the innovation of crop germplasm resources.

Key words: STEME, double base editing, directed evolution, germplasm innovation

胡风越, 王克剑. STEME系统：一种助力体内定向进化的新工具[J]. 遗传, 2020, 42(3): 231-235.

Fengyue Hu, Kejian Wang. The STEME system: a novel tool for directed evolution in vivo[J]. Hereditas(Beijing), 2020, 42(3): 231-235.

图/表 1

表1

参考文献 33

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方法	原理	变异类型	体内/体外	优点	缺点
epPCR	基于PCR技术,在PCR反应体系中添加Mg²⁺、Mn²⁺、诱变dNTP类似物^[29],或使用改造的DNA聚合酶^[30],从而引起碱基突变频率和种类的增加	碱基置换	体外	允许高突变率; 易于使用商业配方	氨基酸取代数目十分有限
位点饱和突变	基于PCR技术,以基因序列中特定密码子为目标,使用含有与目标位点核苷酸不匹配的引物混合物,在目标密码子上引入所有可能的密码子的氨基酸替换^[31]	碱基置换	体外	精确定位特定位点,允许多个密码子同时突变,适用已知蛋白的突变	需要掌握足够的结构和生化知识; 密码子冗余增加了后续筛选的范围^[26]
DNA shuffling	利用酶或物理方法将一组含有益突变位点的基因随机酶切成小片段,进行无引物PCR使DNA片段重新组装,最后获得含有多种同源序列的嵌合体^[32]	同源重组	体外	有效积累有益突变;可实现目的蛋白多种特性的共进化	需逐步剔除有害突变;依赖序列同源性
CDE	基于传统的CRISPR/Cas技术,设计覆盖目标基因或特定位点的sgRNAs文库,构建克隆载体,结合转化技术和组织培养,获得突变体库^[5]	插入缺失	体内	原理和操作简单,可广泛运用于植物性状改良或蛋白工程	易造成不必要的蛋白功能丧失或植株死亡
STEME	基于CRISPR碱基编辑技术,设计覆盖目标基因或特定位点的sgRNAs文库,构建克隆载体,结合转化技术和组织培养,获得突变体库^[7]	碱基置换	体内	产物纯度高,提升突变多样性,靶向诱变范围更广泛	编辑效率有待进一步提升

STEME系统：一种助力体内定向进化的新工具

The STEME system: a novel tool for directed evolution in vivo

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[2]	李弘剑，张毅，周天鸿，李月琴. 基因体外随机突变的两种方法的研究[J]. 遗传, 2000, 22(2): 96-100.