CRISPR-Cas9-assisting efficient and sequential genome deletions in Mycobacterium smegmatis

Abstract:

[Background] For its fast growing and non-pathogenic property, Mycobacterium smegmatis is used as a model strain for pathogenic Mycobacterium tuberculosis and a workhorse to produce steroid hormones. But it lacks efficient genome deletion system. [Objective] In this study, we established a high efficiency genome deletion system in M. smegmatis assisted by CRISPR-Cas9. [Methods] The Cas9 backbone plasmid pCas9101 was constructed with a tetracycline-inducible codon-optimized cas9 expression operon. Approximate 1 kb flanked homologous arms and proper gRNA operon were used to construct vectors to study the deletion efficiency on 3β-hydroxysteroid dehydrogenase (MSMEG_5228, 1 071 bp) encoding gene and cholesterol degradation gene cluster (MSMEG_5990-MSMEG_6043, about 48 kb) in M. smegmatis mc2155. The classical p2NIL-pGOAL method with same homologous arms was used as the control to delete the two same DNA segments in M. smegmatis mc2155. Their deletion efficiency were calculated and compared. [Results] When using CRISPR-Cas9 assisted method, 22% and 18% clones showed the expected deletions in MSMEG_5228 gene and cholesterol-degrading gene cluster, respectively. Even the sequential deletion efficiency on both segments can reach 4%. However, no expected deletion mutants were obtained in our experiments with p2NIL-pGOAL method. [Conclusion] This CRISPR-Cas9 assisted system can facilitate genome deletion in M. smegmatis mc2155, and provide a fast and efficient genome manipulation approach for Mycobacterium in the future.