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Background  Targeted therapies in oncology are promising but variants of uncertain significance (VUS) limit their use for clinical management and necessitate functional testing in vitro. Using  BRCA1  and  BRCA2  variants, which have consequences on PARP inhibitor sensitivity, and  POLE  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’ access to targeted therapies.    Methods  We first evaluated the functional impact of 26  BRCA1  and 7  BRCA2  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  POLE  VUS.    Results  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’ 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  POLE  VUS. Among them, two were classified with an intermediate functional impact and one was functionally abnormal. Eventually, four  POLE  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.    Conclusions  Our new rapid functional assay can be used to characterize the functional implication of  BRCA1  and  BRCA2  variants, giving patients whose variants were evaluated as functionally abnormal access to PARP inhibitor treatment. Retrospective analysis of patients’ 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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13073-021-00976-x.

Functional pre-therapeutic evaluation by genome editing of variants of uncertain significance of essential tumor suppressor genes