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Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
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Journal Article
Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria
2023
Overview
Bacterial genome editing commonly relies on chromosomal cleavage with Cas nucleases to counter-select against unedited cells. However, editing normally requires efficient recombination and high transformation efficiencies, which are unavailable in most strains. Here, we show that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive genome editing. Attenuation can be achieved by altering the format or expression strength of guide (g)RNAs; using nucleases with reduced cleavage activity; or engineering attenuated gRNAs (atgRNAs) with disruptive hairpins, perturbed nuclease-binding scaffolds, non-canonical PAMs, or guide mismatches. These modifications greatly increase cell counts and even improve the efficiency of different types of edits for Cas9 and Cas12a in
Escherichia coli
and
Klebsiella oxytoca
. We further apply atgRNAs to restore ampicillin sensitivity in
Klebsiella pneumoniae
, establishing a resistance marker for genetic studies. Attenuating DNA targeting thus offers a counterintuitive means to achieve CRISPR-driven editing across bacteria.
Genome editing in bacteria normally requires efficient recombination and high transformation efficiencies, which often isn’t. Here the authors report that systematically attenuating DNA targeting activity enables RecA-mediated repair in different bacteria, allowing chromosomal cleavage to drive editing.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 45/41
/ 45/44
/ 45/77
/ Bacteria
/ Cleavage
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ DNA
/ E coli
/ Editing
/ Genome, Bacterial - genetics
/ Genomes
/ Humanities and Social Sciences
/ Nuclease
/ Science
