Saturation genome editing of 11 codons and exon 13 of < i > BRCA2 < /i > coupled with chemotherapeutic drug response accurately determines pathogenicity of variants

by Sounak Sahu, Teresa L. Sullivan, Alexander Y. Mitrophanov, M élissa Galloux, Darryl Nousome, Eileen Southon, Dylan Caylor, Arun Prakash Mishra, Christine N. Evans, Michelle E. Clapp, Sandra Burkett, Tyler Malys, Raj Chari, Kajal Biswas, Shyam K. Sharan 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, Breast Cancer gene-2(BRCA2) 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 humanBRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded byBRCA2 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...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research