Outcomes of Genome Targeting in Bacteria Vary between CRISPR Nucleases

The discovery of CRISPR-Cas immune systems in bacteria and archaea has led to many applications based on programmable DNA and RNA targeting with each system’s RNA-guided nucleases. Based on the observation that genome targeting in bacteria leads to cell death, these nucleases have been used to eradicate cells that failed to undergo recombineering as part of gene editing or exhibit virulence or pathogenicity as part of programmable antimicrobials. Previous studies have used individual single-effector nucleases in single strains for these purposes. However, how these nucleases compare across strains remains to be explored. In this study, we compared three single-effector nucleases with different targeting mechanisms, type II-A Cas9, type V-A Cas12a and type VI-A Cas13a, in different bacteria. We initially tested the genome-targeting activity of these nucleases in Escherichia coli MG1655. We found that each nuclease yielded widely varying reductions in the transformation efficiency of a targeting versus non-targeting guide RNA plasmid, with Cas12a yielding the largest reduction. We also identified different mechanisms of escape from targeting, including repair of cleaved DNA by RecA for Cas9 and delayed cell death or recombination within the guide RNA for Cas12a. Finally, we identified distinct means to counter the survival mechanisms, thereby enhancing cell death and reducing escape. These results show that the outcomes of genome targeting with Cas single-effector nucleases in bacteria can vary between nucleases, with implications for the use of CRISPR for genome editing and programmable antimicrobials in bacteria.