Harnessing CRISPR-Cas9 as an anti-mycobacterial system

Abstract

Rapid emergence of drug resistance has posed new challenges to the treatment of mycobacterial infections. As the pace of development of new drugs is slow, alternate treatment approaches are required. Recently, CRISPR-Cas systems have emerged as potential antimicrobials. These sequence-specific nucleases introduce double strand cuts in the target DNA, which if left unrepaired, prove fatal to the host. For most bacteria, homologous recombination repair (HRR) is the only pathway for repair and survival. Mycobacteria is one of the few bacteria which possesses the non-homologous end joining (NHEJ) system in addition to HRR for double strand break repair. To assess the antimicrobial potential of CRISPR-system, Cas9-induced breaks were introduced in the genome of Mycobacterium smegmatis and the survival was studied. While the single strand breaks were efficiently repaired, the organism was unable to repair the double strand breaks efficiently. In a mixed population of antibiotic-resistant and sensitive mycobacterial cells, selectively targeting a factor that confers hygromycin resistance, turned the entire population sensitive to the drug. Further, we demonstrate that the sequence-specific targeting could also be used for curing plasmids from mycobacterium cells. Considering the growing interest in nucleic acid-based therapy to curtail infections and combat antimicrobial resistance, our data shows that CRISPR-systems hold promise for future use as an antimicrobial against drug-resistant mycobacterial infections.