September 21, 2022
Journal Article

Engineering Citrobacter freundii using CRISPR/Cas9 System

Abstract

The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) system is a useful tool to edit genomes quickly and efficiently. However, the use of CRISPR/Cas9 to edit bacterial genomes has been limited to select microbial chassis primarily used for bioproduction of high value products. Thus, expansion of CRISPR/Cas9 tools to other microbial organisms is needed. Here, our aim was to assess the suitability of CRISPR/Cas9 for genome editing of the Citrobacter freundii type strain ATCC 8090. We evaluated the commonly used two plasmid pCas/pTargetF system to enable gene deletions and insertions in C. freundii and determined editing efficiency. The CRISPR/Cas9 based method enabled high editing efficiency (~91%) for deletion of galactokinase (galk) and enabled deletion with various single guide RNA (sgRNA) sequences. To assess the ability of CRISPR/Cas9 tools to insert genes, we used the fluorescent reporter mNeonGreen, an endopeptidase (yebA), and a transcriptional regulator (xylS) and found successful insertion with high efficiency (81-100%) of each gene individually. These results strengthen and expand the use of CRISPR/Cas9 genome editing to C. freundii as an additional microbial chassis.

Published: September 21, 2022

Citation

Alfaro T.D., J.R. Elmore, Z.R. Stromberg, J.R. Hutchison, and B.M. Hess. 2022. Engineering Citrobacter freundii using CRISPR/Cas9 System. Journal of Microbiological Methods 200. PNNL-SA-173650. doi:10.1016/j.mimet.2022.106533

Research topics