An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition

Abstract

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 <i>Enterococcus faecalis</i>, since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal <i>E. faecalis</i> strains, the genomes of multidrug-resistant (MDR) <i>E. faecalis</i> clinical isolates are enriched for mobile genetic elements (MGEs) and lack <u>c</u>lustered <u>r</u>egularly <u>i</u>nterspaced <u>s</u>hort <u>p</u>alindromic <u>r</u>epeats (CRISPR) and <u>C</u>RISPR-<u>as</u>sociated protein (Cas) genome defense systems. CRISPR-Cas systems cleave foreign DNA in a programmable, sequence-specific manner and are disadvantageous for MGE-derived genome expansion. An unexplored facet of CRISPR biology in <i>E. faecalis</i> is that MGEs that are targeted by native CRISPR-Cas systems can be maintained transiently. Here, we investigate the basis for this "CRISPR tolerance." We observe that <i>E. faecalis</i> can maintain self-targeting constructs that direct Cas9 to cleave the chromosome, but at a fitness cost. Interestingly, DNA repair genes were not upregulated during self-targeting, but integrated prophages were strongly induced. We determined that low <i>cas9</i> expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that <i>E. faecalis</i> has maximized the potential for DNA acquisition by attenuating its CRISPR machinery, thereby facilitating the acquisition of potentially beneficial MGEs that may otherwise be restricted by genome defense.<b>IMPORTANCE</b> 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 <i>Enterococcus faecalis</i> is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in <i>E. faecalis</i> 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.