Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Cancer Research

Background:

• Traditional methods for assessing BRCA1 gene mutations, such as reporter assays and stem cell viability assays, face limitations including overexpression artifacts and inability to address post-transcriptional regulation.
• Accurate functional assessment of BRCA1 variants is crucial for understanding cancer risks and guiding clinical decisions.

Purpose of the Study:

• To develop and present a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editing.
• To overcome the limitations of existing methods for evaluating BRCA1 variants of uncertain significance.

Main Methods:

• Utilized CRISPR-mediated cytosine base editor to induce targeted nucleotide substitutions in living cells for BRCA1 variant analysis.
• Developed a protocol detailing target site selection, functional analysis, and evaluation of BRCA1 variants.
• Applied the method to identify functional impacts of specific ambiguous BRCA1 variants (c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A).

Main Results:

• Demonstrated the utility of CRISPR-based base editors for functional analysis of BRCA1 variants directly within living cells.
• Successfully identified the functional impact of several previously ambiguous BRCA1 variants.
• Confirmed the effectiveness of this novel method for reclassifying variants of uncertain significance in the BRCA1 gene.

Conclusions:

• CRISPR-based cytosine base editing offers a powerful and versatile tool for the functional characterization of BRCA1 variants.
• This protocol facilitates accurate reclassification of BRCA1 variants of uncertain significance, improving genetic risk assessment.
• The method overcomes limitations of exogenous expression assays, providing a more physiologically relevant assessment of variant function.