{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T13:50:06Z","timestamp":1791381006618,"version":"4.3.3"},"update-to":[{"DOI":"10.1371\/journal.pgen.1010940","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2023,9,27]],"date-time":"2023-09-27T00:00:00Z","timestamp":1695772800000}}],"reference-count":71,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2023,9,15]],"date-time":"2023-09-15T00:00:00Z","timestamp":1694736000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"name":"NIH Intramural Research Program"},{"name":"Center for Cancer Research"},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"U.S. National Institutes of Health","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"National Cancer Institute, National Institutes of Health","award":["HHSN261201500003I"],"award-info":[{"award-number":["HHSN261201500003I"]}]}],"content-domain":{"domain":["www.plosgenetics.org"],"crossmark-restriction":false},"short-container-title":["PLoS Genet"],"abstract":"<jats:p>The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, <jats:italic>Breast Cancer gene-2 (BRCA2)<\/jats:italic> has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human <jats:italic>BRCA2<\/jats:italic> for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by <jats:italic>BRCA2<\/jats:italic> exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 <jats:italic>BRCA2<\/jats:italic> variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of <jats:italic>BRCA2<\/jats:italic> VUS.<\/jats:p>","DOI":"10.1371\/journal.pgen.1010940","type":"journal-article","created":{"date-parts":[[2023,9,15]],"date-time":"2023-09-15T17:38:12Z","timestamp":1694799492000},"page":"e1010940","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgen.corrections_policy","source":"Crossref","is-referenced-by-count":23,"title":["Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants"],"prefix":"10.1371","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7290-6651","authenticated-orcid":true,"given":"Sounak","family":"Sahu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teresa L.","family":"Sullivan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9019-4034","authenticated-orcid":true,"given":"Alexander 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