Portable CRISPR-Cas9 N System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei

This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species.