CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.