An Improved Method to Edit Undomesticated Lactobacilli Strains Using Crispr-Cas9 Systems

The rise in new genetic tools have given scientists powerful ways to modify and study prokaryotes. However, many of these methods require a high transformation efficiency of the bacterial strains in order to work, which significantly limits use of these methods across species. Here, we developed a novel method utilizing CRISPR-Cas9 and repair templates to genetically edit low transformation efficient strain of abundant Lactobacillus Plantarum. By using CRISPR-Cas9 in these strains, we show that single point mutations are almost impossible to achieve without using a double stranded DNA repair template. We plan to use this method to further probe and study the diverse lactobacilli genus. This method offers a powerful means of editing lactobacilli and should be translatable to other strains of difficult to work with bacteria.