基于 CRISPR-Cas12a 的基因组编辑和转录抑制在变色假单胞菌生物素合成中的应用。

CRISPR-Cas12a-based genome editing and transcriptional repression for biotin synthesis in Pseudomonas mutabilis.

机构信息

Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.

Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.

出版信息

J Appl Microbiol. 2023 Mar 1;134(3). doi: 10.1093/jambio/lxad049.

DOI:10.1093/jambio/lxad049

PMID:36914213

Abstract

AIMS

To establish a dual-function clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system combined genome editing and transcriptional repression for multiplex metabolic engineering of Pseudomonas mutabilis.

MATERIALS AND RESULTS

This CRISPR-Cas12a system consisted of two plasmids that enabled single gene deletion, replacement, and inactivation with efficiency >90% for most targets within 5 days. With the guidance of truncated crRNA containing 16 bp spacer sequences, a catalytically active Cas12a could be employed to repress the expression of the reporter gene eGFP up to 66.6%. When bdhA deletion and eGFP repression were tested simultaneously by transforming a single crRNA plasmid and Cas12a plasmid, the knockout efficiency reached 77.8% and the expression of eGFP was decreased by >50%. Finally, the dual-functional system was demonstrated to increase the production of biotin by 3.84-fold, with yigM deletion and birA repression achieved simultaneously.

CONCLUSIONS

This CRISPR-Cas12a system is an efficient genome editing and regulation tool to facilitate the construction of P. mutabilis cell factories.

摘要

目的

建立一种双功能成簇规律间隔短回文重复（CRISPR）-Cas12a 系统，结合基因组编辑和转录抑制，用于灵活假单胞菌的多代谢工程。

材料与结果

该 CRISPR-Cas12a 系统由两个质粒组成，可在 5 天内实现大多数目标的基因缺失、替换和失活，效率>90%。在含有 16 个碱基间隔序列的截断 crRNA 的指导下，具有催化活性的 Cas12a 可将报告基因 eGFP 的表达抑制高达 66.6%。当通过转化单个 crRNA 质粒和 Cas12a 质粒同时测试 bdhA 缺失和 eGFP 抑制时，敲除效率达到 77.8%，eGFP 的表达降低了>50%。最后，该双功能系统被证明可将生物素的产量提高 3.84 倍，同时实现 yigM 缺失和 birA 抑制。