Genome Editing with Cas9 in Lactobacilli

The bacterial genus Lactobacillus comprises a vast range of strains with varying metabolic and probiotic traits, with genome editing representing an essential tool to probe genotype–phenotype relationships and enhance their beneficial properties. Currently, one of the most effective means of genome editing in bacteria couples low-efficiency recombineering with high-efficiency counterselection by nucleases from CRISPR-Cas systems. In lactobacilli, several CRISPR-based genome editing methods exist that have shown varying success in different strains. Here, we detail a fast and simple approach using two shuttle vectors encoding a recombineering template as well as the Streptococcus pyogenes Cas9, a trans-activating RNA, and a CRISPR array. We provide a step-by-step procedure for cloning the shuttle vectors, sequentially transforming the vectors into lactobacilli, screening for the desired edit, and finally clearing the shuttle vectors from the mutant strain. As CRISPR-based genome editing in bacteria can fail for various reasons, we also lay out instructions for probing mechanisms of escape. Finally, we include practical notes along the way to facilitate each stage of genome editing, and we illustrate the technique using a representative edit in a strain of Lactobacillus plantarum. Overall, this method should serve as a complete guide to performing genome editing in lactobacilli.