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

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. Competing Interest Statement The authors have declared no competing interest. Footnotes Corrected error in author EJG's middle initial.