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Using<jats:italic>BRCA1<\/jats:italic>and<jats:italic>BRCA2<\/jats:italic>variants, which have consequences on PARP inhibitor sensitivity, and<jats:italic>POLE<\/jats:italic>variants, potential biomarkers of immunotherapy response, we developed a rapid functional assay based on CRISPR-Cas9 genome editing to determine the functional consequences of these variants having potentially direct implications on patients\u2019 access to targeted therapies.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We first evaluated the functional impact of 26<jats:italic>BRCA1<\/jats:italic>and 7<jats:italic>BRCA2<\/jats:italic>variants by editing and comparing NGS results between the variant of interest and a silent control variant. Ten of these variants had already been classified as benign or pathogenic and were used as controls. Finally, we extended this method to the characterization of<jats:italic>POLE<\/jats:italic>VUS.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>For the 23 variants that were unclassified or for which conflicting interpretations had been reported, 15 were classified as functionally normal and 6 as functionally abnormal. Another two variants were found to have intermediate consequences, both with potential impacts on splicing. We then compared these scores to the patients\u2019 responses to PARP inhibitors when possible. Finally, to prove the application of our method to the classification of variants from other tumor suppressor genes, we exemplified with three<jats:italic>POLE<\/jats:italic>VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four<jats:italic>POLE<\/jats:italic>variants previously classified in databases were also evaluated. However, we found evidence of a discordance with the classification, variant p.Leu424Val being found here functionally normal.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>Our new rapid functional assay can be used to characterize the functional implication of<jats:italic>BRCA1<\/jats:italic>and<jats:italic>BRCA2<\/jats:italic>variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients\u2019 responses to PARP inhibitors, where accessible, was consistent with our functional score evaluation and confirmed the accuracy of our protocol. This method could potentially be extended to the classification of VUS from all essential tumor suppressor genes and can be performed within a timeframe compatible with clinical applications, thereby having a direct theranostic impact.<\/jats:p><\/jats:sec>","DOI":"10.1186\/s13073-021-00976-x","type":"journal-article","created":{"date-parts":[[2021,11,9]],"date-time":"2021-11-09T00:02:27Z","timestamp":1636416147000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes"],"prefix":"10.1186","volume":"13","author":[{"given":"Amandine","family":"Billaud","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Louise-Marie","family":"Chevalier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paule","family":"Augereau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jean-Sebastien","family":"Frenel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christophe","family":"Passot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Campone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0125-1883","authenticated-orcid":false,"given":"Alain","family":"Morel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,11,9]]},"reference":[{"issue":"7187","key":"976_CR1","doi-asserted-by":"publisher","first-page":"564","DOI":"10.1038\/nature06915","volume":"452","author":"LJ Van\u2019t Veer","year":"2008","unstructured":"Van\u2019t Veer LJ, Bernards R. 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