Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Automated summary

This study presents a new method for functional analysis of BRCA1 variants using CRISPR-mediated cytosine base editor, overcoming issues related to overexpression and post-transcriptional regulation. The researchers identified ambiguous BRCA1 variants and demonstrated the effectiveness of CRISPR-mediated base editors in reclassifying variants of uncertain significance.