Engineering Citrobacter freundii using CRISPR/Cas9 system

Alfaro, Trinidad D.; Elmore, Joshua R.; Stromberg, Zachary R.; Hutchison, Janine R.; Hess, Becky M.

doi:10.1016/j.mimet.2022.106533

Engineering Citrobacter freundii using CRISPR/Cas9 system

Journal Article · Thu Jun 30 00:00:00 EDT 2022 · Journal of Microbiological Methods

DOI:https://doi.org/10.1016/j.mimet.2022.106533· OSTI ID:1885753

Alfaro, Trinidad D. ^[1]; Elmore, Joshua R. ^[1]; Stromberg, Zachary R. ^[1]; Hutchison, Janine R. ^[1]; Hess, Becky M. ^[1]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

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.

View Accepted Manuscript (DOE)

Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE; US Office of the Director of National Intelligence (ODNI), Intelligence Advanced Research Projects Activity (IARPA)

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1885753

Report Number(s):: PNNL-SA-173650

Journal Information:: Journal of Microbiological Methods, Journal Name: Journal of Microbiological Methods Vol. 200; ISSN 0167-7012

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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