Hillebrand G H, Carlin S C, Giacobe E J, Stephenson H A, Collins J, Hooven T A
Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh PA, USA.
Department of Pediatrics, Division of Newborn Medicine, University of Pittsburgh School of Medicine, Pittsburgh PA, USA.
bioRxiv. 2025 Jun 24:2025.06.20.660720. doi: 10.1101/2025.06.20.660720.
(group B ; 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 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.
B组链球菌（GBS）是新生儿败血症和脑膜炎的主要病因。尽管分子微生物学取得了进展，但由于诱变方案效率低下，GBS基因组工程仍然很费力。在这里，我们报告了一种用于GBS基因操作的通用且快速的基于Cas12a的工具包。我们开发了两种穿梭质粒——用于基因组编辑的pGBSedit和用于诱导型CRISPR干扰的pGBScrispri，它们源自一个系统并针对GBS进行了优化。使用这些工具，我们通过替代末端连接修复实现了靶向基因插入、无标记缺失以及高效的无模板诱变。此外，一种催化失活的dCas12a变体实现了诱导型基因沉默，并具有链特异性靶向效应。全基因组测序证实，该系统在多种GBS菌株中具有广泛的适用性且脱靶活性极低。这个基于Cas12a的平台为GBS的基因研究提供了一种快速、灵活且可扩展的方法，有助于功能基因组学研究并加速发病机制研究。
