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

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 human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 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 BRCA2 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 BRCA2 VUS.Author summary: The exponential rise in genetic sequencing led to the identification of several missense variants. However, its clinical utility is often limited by our ability to determine the functional impact of genetic variants. This has resulted in the identification of many variant of uncertain significance (VUS), particularly in BRCA2. Several functional assays have been developed to ascertain the impact of VUSs on protein function that can be used to determine their pathogenicity. Our mouse ES cell (mESC)-based method includes generation of individual BRCA2 variants in a bacterial artificial chromosome (BAC) by recombineering and assess their expression in mouse ES cells. These cells can then be used to determine how the variants affect mESC viability and sensitivity to DNA damaging agents. Although the assay has high sensitivity and specificity, the process is time consuming and labor-intensive. To overcome these limitations, here we report the development of a CRISPR-Cas9 based high-throughput approach that can be used to classify multiple BRCA2 variants. Specifically, we utilized the BRCA2-independent single-stranded annealing pathway to efficiently knock-in targeted mutations in mESCs using a library of single-stranded DNA donors. Not only do our results support the ClinVar classification of some variants, but we also assign clinical significance to several VUSs, for which no functional data is currently available.