Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria

Daphne Collias, Elena Vialetto, Jiaqi Yu, Khoa Co, Éva d. H. Almási, Ann-Sophie Rüttiger, Tatjana Achmedov, Till Strowig and Chase L. Beisel ()
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Daphne Collias: Helmholtz Centre for Infection Research (HZI)
Elena Vialetto: Helmholtz Centre for Infection Research (HZI)
Jiaqi Yu: Helmholtz Centre for Infection Research (HZI)
Khoa Co: Helmholtz Centre for Infection Research (HZI)
Éva d. H. Almási: Helmholtz Centre for Infection Research (HZI)
Ann-Sophie Rüttiger: Helmholtz Centre for Infection Research (HZI)
Tatjana Achmedov: Helmholtz Centre for Infection Research (HZI)
Till Strowig: Helmholtz Centre for Infection Research (HZI)
Chase L. Beisel: Helmholtz Centre for Infection Research (HZI)

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: Abstract 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.

Date: 2023
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DOI: 10.1038/s41467-023-36283-9

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