Tang, D.; Wang, M.-G.; Wang, D.; Yang, D.; Cai, Y.; Luo, T.; He, J.-q.; Wang, Q.

Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 105 CFU per {micro}g DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many non-tuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths, and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency--up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 24-fold in Mycobacterium kansasii--compared to standard conditions (1.25kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools--including expression libraries and CRISPRi knockdown libraries--in species that have historically resisted genetic manipulation.