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The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is strongly associated with late-onset disease due to its unique set of virulence factors. However, genetic manipulation of ST-17 strains remains challenging, limiting the ability to study key pathogenic genes. In this study, we developed a CRISPR interference (CRISPRi) system utilizing an endogenous catalytically inactivated Cas9 (dCas9) in the COH1 strain, enabling targeted and tunable gene expression knockdown. We confirmed the efficacy of this system through hemolysis assays, qPCR transcriptional analysis, and\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      infection models using human brain endothelial cells. The CRISPRi system successfully produced phenotypic knockdowns of key virulence genes, including PI-2b,\n                      <jats:italic toggle=\"yes\">srr2<\/jats:italic>\n                      , and\n                      <jats:italic toggle=\"yes\">iagA<\/jats:italic>\n                      , reducing adhesion, invasion, and inflammatory responses at the blood-brain barrier (BBB). This platform enables rapid gene knockdowns for functional genomics in ST-17 GBS, enabling high-throughput screening and pathogenesis research.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>Group B Streptococcus (GBS) remains the world's leading cause of neonatal meningitis. GBS-host interactions at the blood-brain barrier (BBB) are dependent on bacterial factors, including surface factors and two-component systems. Multi-locus sequence type 17 (ST-17) GBS strains are highly associated with neonatal meningitis, and these strains harbor many virulence factors for infection at the BBB. Historically, these factors have been studied using traditional knockout mutagenesis, which has been challenging in the most common ST-17 lab strain, COH1. This study utilizes CRISPR interference (CRISPRi) to generate rapid expression knockdown. This study validates a CRISPRi-enabled COH1 dCas9 strain as a versatile tool for probing GBS pathogenesis at the BBB.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jb.00376-25","type":"journal-article","created":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T14:00:20Z","timestamp":1768399220000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["CRISPR interference in a\n                    <i>Streptococcus agalactiae<\/i>\n                    multi-locus sequence type 17 strain"],"prefix":"10.1128","volume":"208","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7881-2127","authenticated-orcid":true,"given":"William D.","family":"Cutts","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/049emcs32","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Molecular and Cell Biology, University of Texas at Dallas","place":["Dallas, 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