An adapted method for Cas9-mediated editing reveals the species-specific role of ß-glucoside utilization driving competition between Klebsiella species.

Cas9-based gene editing tools have revolutionized genetics, enabling the fast and precise manipulation of diverse bacterial species. However, widely applicable genetic tools for non-model gut bacteria are unavailable. Here, we present a two-plasmid Cas9-based system designed for gene deletion and knock-in complementation in three members of the Klebsiella oxytoca species complex (KoSC), which we applied to study the genetic factors underlying the role of these bacteria in competition against Klebsiella pneumoniae. Firstly, the system allowed efficient and precise full-length gene deletion via enhanced lambda Red expression. Furthermore, we tested the efficiency of two independent, functionally validated complementation strategies. Ultimately, the insertion of universal "bookmark" targets during gene deletion subsequently allows the most optimal genetic complementation in K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. This approach offers a significant advantage by enabling the use of a single high-efficiency "bookmark" for complementing other loci or strains, eliminating the need for site-specific design. We revealed that the carbohydrate permease CasA is critical in ex vivo assays for K. pneumoniae inhibition by K. oxytoca but is neither sufficient nor required for K. michiganensis and K. grimontii. Thus, the adaptation of state-of-the-art genetic tools to KoSC allows the identification of species-specific functions in microbial competition. IMPORTANCE: Cas9-based gene editing tools have revolutionized bacterial genetics, yet, their application to non-model gut bacteria is frequently hampered by various limitations. We utilized a two-plasmid Cas9-based system designed for gene deletion in Klebsiella pneumoniae and demonstrate after optimization its utility for gene editing in three members of the Klebsiella oxytoca species complex (KoSC) namely K. oxytoca, Klebsiella michiganensis, and Klebsiella grimontii. We then adapted a recently developed protocol for functional complementation based on universal "bookmark" targets applicable to all tested species. In summary, species-specific adaptation of state-of-the-art genetic tools allows efficient gene deletion and complementation in type strains as well as natural isolates of KoSC members to study microbial interactions.

Systematically attenuating DNA targeting enables CRISPR-driven editing in bacteria.

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.