NT-CRISPR: Combining natural transformation and CRISPR/Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for a wide range of projects. To fully harness the potential of this fascinating bacterium, convenient and highly efficient genome editing methods are indispensable to create novel strains, tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This process, called natural transformation, was tamed for genome editing. It displays a high efficiency and is able to mediate uptake of multiple DNA fragments, thereby allowing multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR/Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR/Cas9, targeting the wild type sequence, leading to death of non-edited cells. Through highly efficient cell killing with efficiencies of up to 99.999 %, integration of antibiotic resistance markers became dispensable and thus enabled scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100 % and further demonstrated its applicability for the simultaneous deletion of multiple chromosomal regions. Lastly, we demonstrated that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby massively broadens the target spectrum.