{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T06:02:43Z","timestamp":1781071363641,"version":"3.54.1"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T00:00:00Z","timestamp":1781049600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Methicillin-Resistant\n                      <jats:italic>Staphylococcus aureus<\/jats:italic>\n                      (MRSA) is a major clinical challenge due to its biofilm-forming ability. Innovative therapeutic strategies are essential to prevent bacterial attachment and disrupt biofilm structures. This study investigates the potential of CRISPR technology as a tool to combat resistance by targeting the biofilm-associated genes\n                      <jats:italic>icaA<\/jats:italic>\n                      ,\n                      <jats:italic>icaD<\/jats:italic>\n                      , and\n                      <jats:italic>bap<\/jats:italic>\n                      in MRSA.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Specific guide RNAs were cloned into pCasSA plasmids to target the\n                      <jats:italic>icaA<\/jats:italic>\n                      ,\n                      <jats:italic>icaD<\/jats:italic>\n                      , and\n                      <jats:italic>bap<\/jats:italic>\n                      genes. Gene expression changes were quantified using quantitative PCR (qPCR), and mutations were confirmed through Sanger sequencing. Biofilm formation was assessed by crystal violet assay, and antimicrobial susceptibility was evaluated by broth microdilution and disk diffusion methods.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      qPCR analyses confirmed significant reductions in gene expression: 3.3-fold for\n                      <jats:italic>icaA<\/jats:italic>\n                      , 2.3-fold for\n                      <jats:italic>icaD<\/jats:italic>\n                      , and 1.7-fold for\n                      <jats:italic>bap<\/jats:italic>\n                      . Sanger sequencing confirmed point mutations and indels within the target regions of genes. Biofilm formation decreased markedly, with 6-fold in icaA-mutant, 5.6-fold in icaD-mutant, and threefold in bap-mutant strains. MIC values were substantially reduced in all mutant strains: oxacillin MIC decreased 64-fold, 16-fold, and 4-fold in\n                      <jats:italic>icaA<\/jats:italic>\n                      -,\n                      <jats:italic>icaD<\/jats:italic>\n                      -, and\n                      <jats:italic>bap<\/jats:italic>\n                      -mutants, respectively, and ciprofloxacin MIC decreased 64\u2013128-fold. Zone diameters for cefoxitin, norfloxacin, and gentamicin increased up to twofold across all mutant strains. All strains remained resistant according to EUCAST clinical breakpoints.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>\n                      This study demonstrates that CRISPR-Cas9-mediated disruption of biofilm-associated genes is an effective strategy for inhibiting biofilm formation in MRSA. Targeted disruption of\n                      <jats:italic>icaA<\/jats:italic>\n                      ,\n                      <jats:italic>icaD<\/jats:italic>\n                      , and\n                      <jats:italic>bap<\/jats:italic>\n                      significantly reduced biofilm formation and partially attenuated antimicrobial resistance phenotypes. These findings highlight the potential of pathogen-specific CRISPR-based anti-virulence strategies as complementary approaches for the treatment of biofilm-associated infections.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1843899","type":"journal-article","created":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T05:36:56Z","timestamp":1781069816000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Targeting biofilm-related genes in a clinical methicillin-resistant Staphylococcus aureus isolate using CRISPR-Cas9 gene editing"],"prefix":"10.3389","volume":"17","author":[{"given":"Aysegul","family":"Ates","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ege","place":["Izmir, T\u00fcrkiye"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sohret","family":"Aydemir","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, University of Ege","place":["Izmir, T\u00fcrkiye"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Safak","family":"Ermertcan","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Ege","place":["Izmir, T\u00fcrkiye"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,6,10]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"7641","DOI":"10.2147\/IDR.S428103","article-title":"Methicillin resistant Staphylococcus aureus: molecular mechanisms underlying drug resistance development and novel strategies to combat","volume":"16","author":"Abebe","year":"2023","journal-title":"Infect Drug Resist"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"261","DOI":"10.3390\/cells13030261","article-title":"Systematic comparison of computational tools for sanger sequencing-based genome editing analysis","volume":"13","author":"Aoki","year":"2024","journal-title":"Cells"},{"key":"ref3","doi-asserted-by":"publisher","first-page":"1329","DOI":"10.32592\/ARI.2024.79.6.1329","article-title":"Evaluation of icaA and icaD genes involved in biofilm formation in Staphylococcus aureus isolates from clinical sources using reverse transcriptase PCR","volume":"79","author":"Armoon","year":"2024","journal-title":"Arc Razi Inst"},{"key":"ref4","doi-asserted-by":"publisher","first-page":"5061","DOI":"10.2147\/IDR.S379502","article-title":"Biofilm-associated multi-drug resistance in hospital-acquired infections: a review","volume":"15","author":"Assefa","year":"2022","journal-title":"Infect Drug Resist"},{"key":"ref5","doi-asserted-by":"publisher","first-page":"374","DOI":"10.1089\/crispr.2024.0001","article-title":"CRISPR-Cas9-mediated targeting of multidrug resistance genes in methicillin-resistant Staphylococcus aureus","volume":"7","author":"Ates","year":"2024","journal-title":"CRISPR J"},{"key":"ref6","doi-asserted-by":"publisher","first-page":"2545","DOI":"10.22207\/JPAM.19.4.13","article-title":"Targeting biofilm-forming Staphylococcus aureus in healthcare: innovations in prevention, eradication, and resistance management","volume":"19","author":"Badge","year":"2025","journal-title":"J Pure Appl Microbiol."},{"key":"ref7","doi-asserted-by":"publisher","first-page":"101147","DOI":"10.1016\/j.drup.2024.101147","article-title":"Mechanism of staphylococcal resistance to clinically relevant antibiotics","volume":"77","author":"Brdov\u00e1","year":"2024","journal-title":"Drug Resist"},{"key":"ref8","year":"2019"},{"key":"ref9","doi-asserted-by":"publisher","first-page":"246","DOI":"10.1186\/s40001-024-01831-6","article-title":"Unveilling genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance in Staphylococcus aureus isolated from a Malaysian teaching hospital","volume":"29","author":"Chan","year":"2024","journal-title":"Eur. 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