Disrupting Homologous Recombination or Single-Strand Annealing Significantly Hinders CRISPR-Cas12a-Assisted Nonhomologous End-Joining Gene Editing Efficiency in Mycobacterium abscessus

Mycobacterium abscessus , a fast-growing, non-tuberculous mycobacterium resistant to most antimicrobial drugs, poses a significant public health challenge because of its ability to cause many types of serious infections in humans. While genetic manipulation tools for M. abscessus are still being developed, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems have shown promise in generating highly specific double-strand breaks (DSBs) in its genome. These DSBs can be repaired by the error-prone nonhomologous end joining (NHEJ) mechanism, facilitating targeted gene editing. Here, our study marks a pioneering application of the CRISPR-NHEJ strategy in M. abscessus . Moreover, our research uncovered an unexpected finding: contrary to previous observations in Mycobacterium tuberculosis and other species, the inhibition of RecA or disruption of key genes in the homologous recombination or single-strand annealing pathways resulted in a significant decrease in NHEJ repair efficiency in M. abscessus . This discovery challenges the established perspectives and offers new insights into the interaction among the three DSB repair pathways in Mycobacterium species.