{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:06:38Z","timestamp":1785420398910,"version":"3.56.0"},"posted":{"date-parts":[[2025,6,21]]},"group-title":"Microbiology","reference-count":51,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,6,21]],"date-time":"2025-06-21T00:00:00Z","timestamp":1750464000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R21AI178067"],"award-info":[{"award-number":["R21AI178067"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01AI182835"],"award-info":[{"award-number":["R01AI182835"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01AI177991"],"award-info":[{"award-number":["R01AI177991"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Centers of Biomedical Research Excellence","award":["P20GM125504"],"award-info":[{"award-number":["P20GM125504"]}]},{"name":"UPMC Children's Hospital of Pittsburgh Research Advisory Council","award":["Graduate Student Grant (GHH)"],"award-info":[{"award-number":["Graduate Student Grant (GHH)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,6,24]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  <jats:italic>Streptococcus agalactiae<\/jats:italic>\n                  (group B\n                  <jats:italic>Streptococcus<\/jats:italic>\n                  ; 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\u2014pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference\u2014derived from an\n                  <jats:italic>Enterococcus faecium<\/jats:italic>\n                  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.\n                <\/jats:p>","DOI":"10.1101\/2025.06.20.660720","type":"posted-content","created":{"date-parts":[[2025,6,22]],"date-time":"2025-06-22T01:10:10Z","timestamp":1750554610000},"source":"Crossref","is-referenced-by-count":1,"title":["A Cas12a Toolbox for Rapid and Flexible Group B\n                  <i>Streptococcus<\/i>\n                  Genomic Editing and CRISPRi"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3391-4306","authenticated-orcid":false,"given":"GH","family":"Hillebrand","sequence":"first","affiliation":[{"name":"Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7455-5127","authenticated-orcid":false,"given":"SC","family":"Carlin","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Newborn Medicine, University of Pittsburgh School of Medicine, Pittsburgh PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-8506-011X","authenticated-orcid":false,"given":"EJ","family":"Giacobe","sequence":"additional","affiliation":[{"name":"Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"HA","family":"Stephenson","sequence":"additional","affiliation":[{"name":"Dietrich School of Arts and Sciences, University of Pittsburgh, Pittsburgh PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J","family":"Collins","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1959-186X","authenticated-orcid":false,"given":"TA","family":"Hooven","sequence":"additional","affiliation":[{"name":"Program in Microbiology and Immunology, University of Pittsburgh School of Medicine, Pittsburgh PA, USA"},{"name":"Department of Pediatrics, Division of Newborn Medicine, University of Pittsburgh School of Medicine, Pittsburgh PA, USA"},{"name":"RK Mellon Institute for Pediatric Research, Pittsburgh PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041209564415000_2025.06.20.660720v2.1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cix653"},{"key":"2026041209564415000_2025.06.20.660720v2.2","doi-asserted-by":"publisher","DOI":"10.1016\/S2214-109X(22)00093-6"},{"key":"2026041209564415000_2025.06.20.660720v2.3","doi-asserted-by":"publisher","DOI":"10.3389\/fped.2018.00027"},{"key":"2026041209564415000_2025.06.20.660720v2.4","doi-asserted-by":"crossref","unstructured":"Brokaw A , Furuta A , Dacanay M , Rajagopal L , Waldorf KMA . 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