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 de

SUBMITTER: Hillebrand GH 

PROVIDER: S-EPMC12588400 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature