Streamlined, recombinase-free genome editing with CRISPR-Cas9 inLactobacillus plantarumreveals barriers to efficient editing

Abstract

ABSTRACTLactic-acid bacteria such asLactobacillus plantarumare commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome-editing tools are currently being employed to elucidate and enhance these microbes’ beneficial properties. The most advanced tools to-date require heterologous single-stranded DNA recombinases to integrate short oligonucleotides followed by using CRISPR-Cas9 to eliminate cells harboring unedited sequences. Here, we show that encoding the recombineering template on a replicating plasmid allowed efficient genome editing with CRISPR-Cas9 in multipleL. plantarumstrains without a recombinase. This strategy accelerated the genome-editing pipeline and could efficiently introduce a stop codon inribB, silent mutations inackA, and a complete deletion oflacM. In contrast, oligo-mediated recombineering with CRISPR-Cas9 proved far less efficient in at least one instance. We also observed unexpected outcomes of our recombinase-free method, including an ~1.3-kb genomic deletion when targetingribBin one strain, and reversion of a point mutation in the recombineering template in another strain. Our method therefore can streamline targeted genome editing in different strains ofL. plantarum, although the best means of achieving efficient editing may vary based on the selected sequence modification, gene, and strain.