Antibiotic resistant bacteria are critical public health concerns. Among the prime causative factors for the spread of antibiotic resistance is horizontal gene transfer (HGT). A useful model organism for investigating the relationship between HGT and antibiotic resistance is the opportunistic pathogen Enterococcus faecalis, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal E. faecalis strains, the genomes of multidrug-resistant (MDR) E. faecalis clinical isolates are enriched for mobile genetic elements (MGEs) and lack CRISPR-Cas genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. A unique facet of CRISPR biology in E. faecalis is that MGEs that are targeted by native CRISPR-Cas systems can be transiently maintained. Here, we investigate the basis for this “CRISPR tolerance." We observe that E. faecalis can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, no canonical DNA damage response was observed during selftargeting, but integrated prophages were strongly induced. We determined that low cas9 expression is the genetic basis for this transient non-lethality and use this knowledge to develop a robust genome editing scheme. Our discovery of a semi-lethal CRISPR-Cas system suggests that E. faecalis has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.

CRISPR-Cas has provided a powerful toolkit to manipulate bacteria, resulting in improved genetic manipulations and novel antimicrobials. These powerful applications rely on the premise that CRISPR-Cas chromosome targeting, which leads to double-stranded DNA breaks, is lethal. In this study, we show that chromosomal CRISPR targeting in Enterococcus faecalis is transiently non-lethal, the first demonstration of such a phenomenon. We uncover novel phenotypes associated with this “CRISPR tolerance” and, after determining its genetic basis, develop a genome editing platform in E. faecalis with negligible off-target effects. Our findings reveal a novel strategy exploited by a bacterial pathogen to cope with CRISPR-induced conflicts to more readily accept DNA, and our robust CRISPR editing platform will help simplify genetic modifications in this organism.