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

• Published in: mBio 2018-05, Vol.9 (3)
• Main Authors: Hullahalli, Karthik, Rodrigues, Marinelle, Nguyen, Uyen Thy, Palmer, Kelli
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR-Cas; CRISPR-Cas Systems; DNA Replication; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Enterococcus faecalis; Enterococcus faecalis - genetics; Enterococcus faecalis - metabolism; genome editing; horizontal gene transfer
• ISSN: 2161-2129; 2150-7511
• DOI: 10.1128/mBio.00414-18

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 , since the species possesses highly conjugative plasmids that readily disseminate antibiotic resistance genes and virulence factors in nature. Unlike many commensal strains, the genomes of multidrug-resistant (MDR) clinical isolates are enriched for mobile genetic elements (MGEs) and lack lustered egularly nterspaced hort alindromic epeats (CRISPR) and RISPR- 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 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 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 expression contributes to this transient nonlethality and used this knowledge to develop a robust CRISPR-assisted genome-editing scheme. Our results suggest that 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. 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 is transiently nonlethal. We uncover novel phenotypes associated with this "CRISPR tolerance" and, after determining its genetic basis, develop a genome-editing platform in 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.