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The tetraspanins CD9, CD81, and CD63 are crucial to EV biogenesis and function. This study uses CRISPR\u2010Cas9 system to knock out (KO) CD9, CD63, and CD81 in HEK293T cells. The goal is to investigate the role of these tetraspanins in EV bioengineering with the hypothesis that repressing endogenous production may increase the availability of exogenously introduced tetraspanin\u2010fusion constructs and increase engineered EV production. First, it is observed that individually knocking out a tetraspanin does not significantly affect EV formation. However, when all three tetraspanins are simultaneously knocked out, there is a marked decrease in EV production, as measured by nanoparticle tracking analysis. Second, upon reintroduction of the corresponding tetraspanins fused to firefly ThermoLuc or neon green into the PanKO\u2010, CD9KO\u2010, CD63KO\u2010, and CD81KO\u2010cells, the engineered EVs display a significant increase in production by 50% to 70% compared to transduction of wild\u2010type cells, as measured by luminometer and imaging flow cytometry. These findings emphasize the potential of tetraspanin KO in the bioengineering of EVs, paving the way for new therapeutic applications by enhancing production and potentially modifying their cargo.<\/jats:p>","DOI":"10.1002\/smsc.202500533","type":"journal-article","created":{"date-parts":[[2026,3,24]],"date-time":"2026-03-24T15:50:21Z","timestamp":1774367421000},"source":"Crossref","is-referenced-by-count":1,"title":["Evaluation of Tetraspanins in Extracellular Vesicle Bioengineering"],"prefix":"10.1002","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4785-0796","authenticated-orcid":false,"given":"Doste R.","family":"Mamand","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine Division of Biomolecular and Cellular Medicine Karolinska Institute  Stockholm Sweden"},{"name":"Breast Center Karolinska Comprehensive Cancer Center Karolinska University Hospital  Stockholm Sweden"},{"name":"Biology Research center Research Center University of Zakho  Duhok Kurdistan Region Iraq"},{"name":"Karolinska ATMP Center ANA Futura  Huddinge Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Oskar","family":"Gustafsson","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine Division of Biomolecular and Cellular Medicine Karolinska Institute  Stockholm Sweden"},{"name":"Karolinska ATMP Center ANA Futura  Huddinge Sweden"},{"name":"Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST) Karolinska University Hospital  Huddinge Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helena","family":"Sork","sequence":"additional","affiliation":[{"name":"Institute of Technology University of Tartu  Tartu Estonia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rim Jawad","family":"Wiklander","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine Division of Biomolecular and Cellular Medicine Karolinska Institute  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Safa","family":"Bazaz","sequence":"additional","affiliation":[{"name":"Center for Hematology and Regenerative Medicine (HERM) Department of Medicine Huddinge Karolinska Institutet  Stockholm Sweden"},{"name":"Center of Research and Strategic Studies Lebanese French University  Erbil Iraq"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuming","family":"Liang","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine Division of Biomolecular and Cellular Medicine Karolinska Institute  Stockholm Sweden"},{"name":"Karolinska ATMP Center ANA Futura  Huddinge Sweden"},{"name":"Department of Cellular Therapy and Allogeneic Stem Cell Transplantation (CAST) Karolinska University Hospital  Huddinge Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vicky W. 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