Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.

Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.