Functional Analysis of Variants in BRCA1 Using CRISPR Base Editors

Methods Mol Biol. 2023:2606:73-85. doi: 10.1007/978-1-0716-2879-9_7.

Abstract

To date, methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays have been used to evaluate the function of the variants of uncertain significance (VUS) of the BRCA genes. However, these methods have limitations as they are associated with overexpression and do not apply to post-transcriptional regulation. Therefore, there are several VUS whose functions are unclear. Recently, we devised a new way to assess the functionality of variants in BRCA1 via a CRISPR-mediated base editor to overcome these limitations. We precisely introduced the target nucleotide substitution in living cells and identified variants whose functions were not defined. Here, we describe the methods for the functional appraisal of BRCA1 variants using CRISPR-based base editors.

Keywords: Adenine base editor; BRCA1; Base editing; CRISPR; Cytosine base editor; Functional assessment; Genome engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems* / genetics
  • Cytosine
  • Gene Editing* / methods
  • Genes, Tumor Suppressor
  • Nucleotides

Substances

  • Cytosine
  • Nucleotides