Project Description

Nissle 1917 is an E. coli strain with probiotic properties, primarily recognized for its beneficial effects on gastrointestinal health^[1]. However, for certain applications of Nissle 1917, it is necessary to knock out specific genes^[2]. Since CRISPR-Cas systems have high target DNA specificity and programmability, they can serve as gene editing tools^[3]. However, the occurrence of escapers reduces the efficiency of CRISPR-Cas in editing single genes, limiting its application in multiplex editing^[4]. This has become a major obstacle to the development of CRISPR-Cas9-based antimicrobial drugs.

Inspired by Prof. LI QI’s work^[4] , this project aims to construct a highly efficient CRISPR-Cas9-based genome editing tool for E. coli Nissle 1917 by increasing the Cas9 copy number and optimizing the cas9 gene codons, lowering escape rates to improve editing efficiency. Per advice from Prof. Li, we selected higher copy numbers p15A and ColE1 origins of replication and optimized the Cas9 gene codons (Cas9-op).

In this project, we will construct two Cas9 expression plasmids (with optimized Cas9-op and with “old” Cas9), gRNA plasmids, and repair homology arms, and following electroporate into E. coli Nissle 1917 for editing efficiency evaluation.

Custom CRISPR-Cas9-op Editing Kit

We expected that this efficient editing tool can enable the development of customized kits for laboratories requiring gene editing in E. coli Nissle 1917 and will have promising applications in constructing chassis cell lines for various biomanufacturing purposes, increasing target strain yields, and advancing production in diverse biological systems.

Reference

[1] Buddenborg C., Daudel D., Liebrecht S., et al. Development of a tripartite vector system for live oral immunization using a gram-negative probiotic carrier [J]. Int J Med Microbiol, 2008, 298(1-2): 105-114.

[2] Rottinghaus A.G., Ferreiro A., Fishbein S.R.S., et al. Genetically stable crispr-based kill switches for engineered microbes [J]. Nat Commun, 2022, 13(1): 672.

[3] Vento J.M., Crook N., Beisel C.L. Barriers to genome editing with crispr in bacteria [J]. J Ind Microbiol Biotechnol, 2019, 46(9-10): 1327-1341.

[4] Li Q., Sun M., Lv L., et al. Improving the editing efficiency of crispr-cas9 by reducing the generation of escapers based on the surviving mechanism [J]. ACS Synthetic Biology, 2023, 12(3): 672-680.