Deng Chen, Lv Xueqin, Li Jianghua, Liu Yanfeng, Du Guocheng, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.
Metab Eng Commun. 2020 Jun 1;11:e00135. doi: 10.1016/j.mec.2020.e00135. eCollection 2020 Dec.
As a traditional amino acid producing bacterium, is a platform strain for production of various fine chemicals. Based on the CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system, gene editing tools that enable base conversion in the genome of have been developed. However, some problems such as genomic instability caused by DNA double-strand break (DSB) and off-target effects need to be solved. In this study, a DSB-free single nucleotide genome editing system was developed by construction of a bi-directional base conversion tool TadA-dCas9-AID. This system includes cytosine base editors (CBEs): activation-induced cytidine deaminase (AID) and adenine deaminase (ABEs): tRNA adenosine deaminase (TadA), which can specifically target the gene through a 20-nt single guide RNA (sgRNA) and achieve the base conversion of C-T, C-G and A-G in the 28-bp editing window upstream of protospacer adjacent motif. Finally, as a proof-of-concept demonstration, the system was used to construct a mutant library of gene in S9114 genome to improve the production of a typical nutraceutical -acetylglucosamine (GlcNAc). The GlcNAc titer of the mutant strain K293R was increased by 31.9% to 9.1 g/L in shake flask. Here, the developed bases conversion tool TadA-dCas9-AID does not need DNA double-strand break and homologous template, and is effective for genome editing and metabolic engineering in .
作为一种传统的氨基酸生产细菌，是生产各种精细化学品的平台菌株。基于CRISPR（成簇规律间隔短回文重复序列）-Cas9系统，已开发出能够在其基因组中实现碱基转换的基因编辑工具。然而，一些问题，如由DNA双链断裂（DSB）引起的基因组不稳定性和脱靶效应，需要解决。在本研究中，通过构建双向碱基转换工具TadA-dCas9-AID，开发了一种无DSB的单核苷酸基因组编辑系统。该系统包括胞嘧啶碱基编辑器（CBEs）：激活诱导的胞苷脱氨酶（AID）和腺嘌呤脱氨酶（ABEs）：tRNA腺苷脱氨酶（TadA），它们可以通过20个核苷酸的单向导RNA（sgRNA）特异性靶向基因，并在原间隔相邻基序上游的28个碱基对编辑窗口中实现C-T、C-G和A-G的碱基转换。最后，作为概念验证示范，该系统用于在S9114基因组中构建基因的突变文库，以提高典型营养保健品N-乙酰葡萄糖胺（GlcNAc）的产量。在摇瓶中，突变株K293R的GlcNAc滴度提高了31.9%，达到9.1 g/L。在此，开发的碱基转换工具TadA-dCas9-AID不需要DNA双链断裂和同源模板，并且对其基因组编辑和代谢工程有效。
