Saturation genome editing-based clinical classification of BRCA2 variants.

Sequencing-based genetic tests have uncovered a vast array of BRCA2 sequence variants. Owing to limited clinical, familial and epidemiological data, thousands of variants are considered to be variants of uncertain significance (VUS). Here we have utilized CRISPR-Cas9-based saturation genome editing in a humanized mouse embryonic stem cell line to determine the functional effect of VUS.

Characterization of BRCA2 R3052Q variant in mice supports its functional impact as a low-risk variant.

Pathogenic variants in BRCA2 are known to significantly increase the lifetime risk of developing breast and ovarian cancers. Sequencing-based genetic testing has resulted in the identification of thousands of BRCA2 variants that are considered to be variants of uncertain significance (VUS) because the disease risk associated with them is unknown. One such variant is p.

Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing.

Sequencing-based functional assays for classification of BRCA2 variants in mouse ESCs.

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 17,000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUSs).