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Here, we applied a Safe\u2010by\u2010Design strategy using the endogenous CRISPR\u2010Cas system of\n                    <jats:styled-content style=\"fixed-case\">\n                      <jats:italic>B.\u2009animalis<\/jats:italic>\n                    <\/jats:styled-content>\n                    subsp.\n                    <jats:italic>lactis<\/jats:italic>\n                    BLC01 to inactivate\n                    <jats:italic>tetW<\/jats:italic>\n                    through the introduction of premature stop codons. Whole\u2010genome sequencing confirmed the intended editing and excluded relevant off\u2010target effects.\n                    <jats:italic>tetW<\/jats:italic>\n                    inactivation markedly reduced the tetracycline minimum inhibitory concentration, restoring susceptibility below the tetracycline cut\u2010off value for bifidobacteria (8\u2009\u03bcg\/mL). Comparative phenotypic analyses demonstrated that the edited strain (BLC01\u20102F3G10) retained key probiotic traits, including tolerance to acid, bile, and osmotic stress, exopolysaccharide production, aggregation capacity, survival during simulated gastrointestinal digestion and adhesion to intestinal epithelial cells. Importantly, no reversion to tetracycline resistance was observed after prolonged exposure to sub\u2010inhibitory minimal selective antimicrobial concentration, indicating genetic stability of the edited phenotype. Collectively, these findings demonstrate that endogenous CRISPR\u2010based genome editing can be leveraged to selectively remove antimicrobial resistance determinants from probiotic strains while preserving functionality, supporting the development of next\u2010generation probiotics with an improved safety profile and reduced potential for antimicrobial resistance dissemination in the human gut.\n                  <\/jats:p>","DOI":"10.1111\/1751-7915.70443","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T07:21:26Z","timestamp":1789456886000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Endogenous\n                    <scp>CRISPR<\/scp>\n                    \u2010Based Removal of Tetracycline Resistance in\n                    <scp>\n                      <i>Bifidobacterium animalis<\/i>\n                    <\/scp>\n                    subsp.\n                    <scp>\n                      <i>lactis<\/i>\n                    <\/scp>\n                    Through a Safe\u2010by\u2010Design Approach"],"prefix":"10.1111","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-5019-4421","authenticated-orcid":false,"given":"Marianna","family":"Bozzetti","sequence":"first","affiliation":[{"name":"Department for Sustainable Food Process\u2014DiSTAS Universit\u00e0 Cattolica del Sacro Cuore  Cremona Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-1961-2908","authenticated-orcid":false,"given":"Matteo","family":"Raneri","sequence":"additional","affiliation":[{"name":"Sacco srl  Cadorago Como Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6884-091X","authenticated-orcid":false,"given":"Claudia","family":"Cortimiglia","sequence":"additional","affiliation":[{"name":"Department for Sustainable Food Process\u2014DiSTAS Universit\u00e0 Cattolica del 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