CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira

Authors: FERNANDES, LUIS GV - Oak Ridge Institute For Science And Education (ORISE); HAMOND, CAMILA - Diagnostic Virology Laboratory/ National Veterinary Services Laboratories; Tibbs-Cortes, Bienvenido; Putz, Ellie; Olsen, Steven; Palmer, Mitchell; Nally, Jarlath

Submitted to: mBio
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/8/2024
Publication Date: 8/13/2024
Citation: Fernandes, L., Hamond, C., Tibbs-Cortes, B.W., Putz, E.J., Olsen, S.C., Palmer, M.V., Nally, J.E. 2024. CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira. mBio. https://doi.org/10.1128/mbio.01516-24.
DOI: https://doi.org/10.1128/mbio.01516-24

Interpretive Summary: Leptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the L. borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Technical Abstract: Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanism of disease and known virulence factors. The application of CRISPR/Cas9, and its variations, helped filled these gaps but because double-stranded breaks (DSBs) inflicted by Cas9 nuclease is lethal to Leptospira cells, the generation of knockout mutants remains challenging. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletion, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified sgRNA (termed PEgRNA) containing an extended 3’end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate L. borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of marker-less knockout strains, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.