BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发用

doi:10.13560/j.cnki.biotech.bull.1985.2019-0956

生物技术通报 ›› 2020, Vol. 36 ›› Issue (3): 95-101.doi: 10.13560/j.cnki.biotech.bull.1985.2019-0956

• 基因编辑专题（专题主编谷峰研究员） • 上一篇下一篇

BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发用

黄华媚^{1, 2, 3}, 白立宽^{1, 2, 3}, 刘叶^{2, 3}, 李俊维^{1, 2, 3}, 王猛^{2, 3}, 花尔并¹

1. 天津科技大学生物工程学院,天津 300457;
2. 中国科学院系统微生物工程重点实验室,天津 300308;
3. 中国科学院天津工业生物技术研究所,天津 300308

收稿日期:2019-10-10 出版日期:2020-03-26 发布日期:2020-03-17
作者简介:黄华媚,女,硕士研究生,研究方向：生物工程;E-mail：huanghm@tib.cas.cn;白立宽为共同第一作者
基金资助:
国家重点研发计划合成生物学重点专项（2018YFA0902902）,中国科学院前沿科学重点研究项目（QYZDB-SSW-SMC012）,中国科学院战略生物资源计划（KFJ-BRP-009）,中国科学院重点部署项目（KFZD-SW-215）,中国科学院国际合作局对外合作重点项目（153D31KYSB20170121）

Development and Application of BE3 Cytidine Base Editor in Corynebacterium glutamicum

HUANG Hua-mei^{1, 2, 3}, BAI Li-kuan^{1, 2, 3}, LIU Ye^{2, 3}, LI Jun-wei^{1, 2, 3}, WANG Meng^{2, 3}, HUA Er-bing¹

1. College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457;
2. Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308;
3. Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308

Received:2019-10-10 Published:2020-03-26 Online:2020-03-17

摘要/Abstract

摘要： 碱基编辑技术结合了CRISPR/Cas系统的靶向特异性与碱基脱氨酶的催化活性,因其不产生双链DNA断裂、不需要外源DNA模板、不依赖同源重组修复,自开发以来,便受到研究者的追捧,在哺乳动物细胞、植物、微生物等领域相继得到开发与应用。为了进一步丰富碱基编辑系统在谷氨酸棒杆菌中的应用,将鼠源胞嘧啶脱氨酶（rAPOBEC1）与nCas9蛋白融合,实现了在谷氨酸棒杆菌中C到T的编辑,编辑比例较低（0-20%）;在上述融合蛋白C端添加UGI蛋白,构建BE3型胞嘧啶碱基编辑器,抑制体内的DNA碱基切除修复机制,显著的提高了碱基编辑效率,使得C到T的碱基编辑效率高达90%;为了简化操作,将双质粒碱基编辑系统优化为单质粒碱基编辑系统,并显著提高转化效率;最后通过单质粒碱基编辑系统对基因组中其他位点的编辑测试,进一步证明了BE3型碱基编辑器在谷氨酸棒杆菌中的高效性,同时发现该碱基编辑器具有较宽的编辑窗口（PAM上游-11到-19位）,有助于覆盖更多的基因组靶标位点,为谷氨酸棒杆菌的基因组改造提供了更多的工具选择。

关键词: 碱基编辑, 谷氨酸棒杆菌, CRISPR/Cas系统, 胞嘧啶脱氨酶

Abstract: The base editing technology,combining the targeted specificity of CRISPR/Cas system and the catalytic activity of cytidine deaminase,has been developed and widely applied in mammalian cells,plants and microorganism,due to not introducing double-stranded DNA break,a DNA template and relying on host homologous recombination repair pathway. In order to expand the application of base editing in Corynebacterium glutamicum,C to T conversion was achieved by fusing the cytidine deaminase（rAPOBEC1）and nCas9;however,the initial editing efficiency was low（0 to 20%）. The base editor BE3 was then constructed by adding the UGI protein in the C terminus of the rAPOBEC1-nCas9 fusion,which inhibited the DNA base excise repair pathway and significantly improved the editing performance with the C to T conversion efficiency up to 90%. For convenience in future applications,the dual-plasmid base editing system was simplified to single-plasmid system,and the transformation efficiency was remarkably enhanced. Finally,by using other genomic loci as editing target,the single-plasmid based BE3 base editor was proved to be powerful with high editing efficiency and broad editing window（-11 to -19 positions upstream of the PAM sequence）,which was beneficial to cover more target genomic loci and provided more tools for genome engineering of C. glutamicum.

Key words: base editing, Corynebacterium glutamicum, CRISPR/Cas9, cytidine deaminase

黄华媚, 白立宽, 刘叶, 李俊维, 王猛, 花尔并. BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发用[J]. 生物技术通报, 2020, 36(3): 95-101.

HUANG Hua-mei, BAI Li-kuan, LIU Ye, LI Jun-wei, WANG Meng, HUA Er-bing. Development and Application of BE3 Cytidine Base Editor in Corynebacterium glutamicum[J]. Biotechnology Bulletin, 2020, 36(3): 95-101.

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BE3型胞嘧啶碱基编辑器在谷氨酸棒杆菌中的开发用

Development and Application of BE3 Cytidine Base Editor in Corynebacterium glutamicum

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