{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T03:53:55Z","timestamp":1788407635483,"version":"build-2803163510"},"reference-count":53,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2025,9,13]],"date-time":"2025-09-13T00:00:00Z","timestamp":1757721600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI177991"],"award-info":[{"award-number":["R01AI177991"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI182835"],"award-info":[{"award-number":["R01AI182835"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R21AI178067"],"award-info":[{"award-number":["R21AI178067"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Molecular Microbiology"],"published-print":{"date-parts":[[2025,11]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>Streptococcus agalactiae<\/jats:italic>\n                    <\/jats:styled-content>\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\u2010based 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:styled-content style=\"fixed-case\">\n                      <jats:italic>Enterococcus faecium<\/jats:italic>\n                    <\/jats:styled-content>\n                    system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template\u2010free mutagenesis via alternative end\u2010joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand\u2010specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off\u2010target activity, as confirmed by whole\u2010genome sequencing. In benchmarking, template\u2010less Cas12a mutagenesis yielded sequence\u2010confirmed mutants in ~7\u2009days and homology\u2010directed edits in ~7\u201314\u2009days; aTC\u2010resistant colonies arose at ~10\n                    <jats:sup>\u22124<\/jats:sup>\n                    of uninduced CFU, and 27%\u201365% of resistant clones carried the intended homology\u2010directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1\u2009kb arms). These workflows provide a rapid alternative to temperature\u2010sensitive plasmid mutagenesis protocols that typically require \u2265\u20094\u2009weeks. This Cas12a\u2010based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.\n                  <\/jats:p>","DOI":"10.1111\/mmi.70022","type":"journal-article","created":{"date-parts":[[2025,9,13]],"date-time":"2025-09-13T07:06:13Z","timestamp":1757747173000},"page":"449-461","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A Cas12a Toolbox for Rapid and Flexible Group B\n                    <i>Streptococcus<\/i>\n                    Genomic Editing and\n                    <scp>CRISPRi<\/scp>"],"prefix":"10.1111","volume":"124","author":[{"given":"G. H.","family":"Hillebrand","sequence":"first","affiliation":[{"name":"Program in Microbiology and Immunology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S. C.","family":"Carlin","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Division of Newborn Medicine University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"E. J.","family":"Giacobe","sequence":"additional","affiliation":[{"name":"Program in Microbiology and Immunology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"H. A.","family":"Stephenson","sequence":"additional","affiliation":[{"name":"Dietrich School of Arts and Sciences University of Pittsburgh  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2479-5189","authenticated-orcid":false,"given":"J.","family":"Collins","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, School of Medicine University of Louisville  Louisville Kentucky USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1959-186X","authenticated-orcid":false,"given":"T. A.","family":"Hooven","sequence":"additional","affiliation":[{"name":"Program in Microbiology and Immunology University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"},{"name":"Department of Pediatrics, Division of Newborn Medicine University of Pittsburgh School of Medicine  Pittsburgh Pennsylvania USA"},{"name":"RK Mellon Institute for Pediatric Research  Pittsburgh Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,9,13]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-022-02823-7"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.03123"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1089\/cmb.2012.0021"},{"key":"e_1_2_10_5_1","article-title":"Comprehensive and Accurate Genome Analysis at Scale Using DRAGEN Accelerated Algorithms","author":"Behera S.","year":"2024","journal-title":"bioRxiv"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2021.720789"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0906355107"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41570-017-0078"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biotechadv.2016.08.002"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.02427-21"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53439-3"},{"issue":"8","key":"e_1_2_10_12_1","article-title":"Evolutionary Rates and Emergence of Human\u2010Associated GBS Lineages","volume":"7","author":"Cuny C.","year":"2021","journal-title":"Microbial Genomics"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1099824"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1258096"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/148.4.1667"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1128\/aem.63.9.3539-3547.1997"},{"key":"e_1_2_10_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/S2214-109X(22)00093-6"},{"key":"e_1_2_10_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-023-04994-w"},{"key":"e_1_2_10_19_1","doi-asserted-by":"publisher","DOI":"10.1099\/mic.0.052548-0"},{"key":"e_1_2_10_20_1","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.03009-18"},{"key":"e_1_2_10_21_1","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-024-06640-5"},{"key":"e_1_2_10_22_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms15179"},{"key":"e_1_2_10_23_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_2_10_24_1","doi-asserted-by":"publisher","DOI":"10.12688\/f1000research.9363.1"},{"key":"e_1_2_10_25_1","doi-asserted-by":"publisher","DOI":"10.1111\/1348-0421.13015"},{"key":"e_1_2_10_26_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.142.1.165"},{"key":"e_1_2_10_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1923"},{"key":"e_1_2_10_28_1","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cix653"},{"key":"e_1_2_10_29_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12275-021-1012-0"},{"key":"e_1_2_10_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijmm.2014.05.003"},{"key":"e_1_2_10_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2649"},{"key":"e_1_2_10_32_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1009761"},{"key":"e_1_2_10_33_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-021-11243-9"},{"key":"e_1_2_10_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2017.09.007"},{"key":"e_1_2_10_35_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.RA120.012933"},{"key":"e_1_2_10_36_1","doi-asserted-by":"publisher","DOI":"10.1128\/iai.64.9.3818-3826.1996"},{"key":"e_1_2_10_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku623"},{"key":"e_1_2_10_38_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-025-60871-6"},{"key":"e_1_2_10_39_1","doi-asserted-by":"publisher","DOI":"10.3389\/fped.2018.00027"},{"key":"e_1_2_10_40_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.csbj.2022.07.023"},{"key":"e_1_2_10_41_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-019-0131-5"},{"key":"e_1_2_10_42_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.02.022"},{"key":"e_1_2_10_43_1","doi-asserted-by":"publisher","DOI":"10.1093\/infdis\/jiu067"},{"key":"e_1_2_10_44_1","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2164-14-252"},{"key":"e_1_2_10_45_1","doi-asserted-by":"publisher","DOI":"10.1007\/s11033-023-08239-1"},{"issue":"1","key":"e_1_2_10_46_1","first-page":"2460","article-title":"Population Genomics of Group B Streptococcus: Global Structure and Adaptive Evolution","volume":"9","author":"Sheppard S. 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