Application of the ORBIT Protocol for Targeted Gene Deletion to Mycobacterium abscessus

Mycobacterium abscessus (Mab) is an emerging opportunistic pathogen characterized by extensive intrinsic drug resistance, underscoring the need for precise genetic tools to define mechanisms of pathogenicity and persistence. The ORBIT (oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting) system developed by the Sassetti group enables precise gene deletions but has primarily been optimized for Mycobacterium tuberculosis and Mycobacterium smegmatis. Although ORBIT has been used in Mab, here we describe an optimized ORBIT workflow for efficient gene deletion in Mab. We tested key parameters, including plasmid selection, oligonucleotide design, and recombineering conditions. We evaluated oligonucleotide length and purification to improve recovery of targeted mutants and established conditions that enabled reproducible deletion of multiple target genes. Our findings establish a robust and adaptable ORBIT protocol tailored to the genetic characteristics of Mab. This optimized workflow provides a practical framework for precise genetic manipulation and can facilitate functional studies of genes that contribute to Mab physiology, antimicrobial resistance, and pathogenesis.