STEME系统：一种助力体内定向进化的新工具

doi:10.16288/j.yczz.20-041

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

• Frontier Focus • Next Articles

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)

Abstract

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

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

Figures/Tables 1

Table 1

References 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

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

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[1]	Lianchao Tang, Feng Gu. Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM [J]. Hereditas(Beijing), 2020, 42(3): 236-249.
[2]	XIONG Ai-Sheng, YAO Quan-Hong, ZHANG Zhen, PENG Ri-He. Rational Evolutionary Design and Modification of Gene: A Short-path of Direct Evolution [J]. HEREDITAS, 2006, 28(1): 92-96.