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bioRxiv · 10.1101/2025.06.20.660720

A Cas12a Toolbox for Rapid and Flexible Group B Streptococcus Genomic Editing and CRISPRi

Hillebrand, G. H.·Carlin, S. M.·Giacobe, E. L.·Stephenson, H. A.·Collins, J.·Hooven, T. A.
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Abstract

Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids--pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference--derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. This Cas12a-based platform offers a rapid, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.

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Hillebrand, G. H., Carlin, S. M., Giacobe, E. L., Stephenson, H. A., Collins, J., Hooven, T. A.. 2025-06-21. A Cas12a Toolbox for Rapid and Flexible Group B Streptococcus Genomic Editing and CRISPRi. https://doi.org/10.1101/2025.06.20.660720

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