Genome Editing with CRISPR‐Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods

Lactic‐acid bacteria such as Lactobacillus plantarum are commonly used for fermenting foods and as probiotics, where increasingly sophisticated genome‐editing tools are employed to elucidate and enhance these microbes’ beneficial properties. The most advanced tools to date utilize an oligonucleotide or double‐stranded DNA donor for recombineering and Cas9 for targeted DNA cleavage. As the associated methods are often developed in isolation for one strain, it remains unclear how different Cas9‐based editing methods compare across strains. Here, this work directly compares two methods in different strains of L. plantarum : one utilizing a plasmid‐encoded recombineering template and another utilizing an oligonucleotide donor and an inducible DNA recombinase. This comparison reveals one instance in which only the recombineering‐template method generates desired edits and another instance in which only the oligo method generates desired edits. It is further found that both methods exhibit highly variable success editing the same site across multiple L. plantarum strains. Finally, failure modes are identified for the recombineering‐template method, including a consistent genomic deletion and reversion of a point mutation in the recombineering template. This study therefore highlights surprising differences for Cas9‐mediated genome editing between methods and related strains, arguing for the need for multiple, distinct methods when performing CRISPR‐based editing in bacteria.