Co-Expressing the S. Pyogenes Cas9 and Its Tracrrna Induces Potent, Strain-Specific Cytotoxicity in Lactobacilli

The Cas9 nuclease from Streptococcus pyogenes (SpCas9) has proven to be an invaluable tool for genome editing and gene regulation in bacteria. However, accumulating examples have reported cytotoxicity of expressing Cas9 in some bacteria, limiting its use for genome editing. The underlying mechanism of cytotoxicity and how it can be circumvented remains poorly understood. Here, we report a distinct example in which co-expressing SpCas9 and its trans-activating CRISPR RNA (tracrRNA) is heavily cytotoxic in the industrial bacterium Lactobacillus paracasei. We found that transforming a plasmid containing SpCas9 and a tracrRNA resulted in very few colonies in this strain compared to transforming a plasmid containing SpCas9 or the tracrRNA alone. Conversely, this reduction in colonies was not observed when transforming the same plasmid into strains of Lactobacillus plantarum and Lactobacillus rhamnosus. Interestingly, adding a non-targeting crRNA to the plasmid significantly reduced cytotoxicity in L. paracasei. We are currently determining the underlying mechanism by elucidating modes of escape from cytotoxicity, determining the role of DNA nicking and cleavage activity, and identifying putative targets of Cas9. The results of this work are expected to help elucidate the basis of SpCas9 cytotoxicity in bacteria, and in turn help others improve genome editing across bacteria, including industrial relevant Lactobacilli.