Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application of the CRISPR/Cas9 system has facilitated the generation of mutants and subsequent evaluation of phenotypes. Since DNA breaks induced by RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented to overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed to create knockdown mutants, utilizing a catalytically inactive Cas9 (dCas9). Subsequently, the co-expression of CRISPR/Cas9 and a DNA repair system from Mycobacterium smegmatis enabled the generation of scarless knockout mutants. We eliminated plasmids from the lipL32 knockout L. interrogans strain and further achieved multiple gene mutations via gene silencing in this knockout background. Strains lacking both LipL41 and LipL32 and LigA, LigB, and LipL32, were evaluated. The absence of proteins LipL32 and LipL41 had no effect on leptospiral virulence. On the other hand, mutants lacking LigA, LigB, and LipL32 were unable to cause acute disease. The expanded apparatus for genetic manipulation of pathogenic leptospires via the CRISPR/Cas9 system has allowed the evaluation of multiple mutations upon leptospiral virulence. This work shows that LipL32 and LipL41 are not required for acute disease and consolidates LigA and LigB proteins as virulence factors.
Keywords: CRISPR-interference; Leptospira; Lig proteins; LipL32; LipL41; knockdown; knockout; multiple mutations.