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Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

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Abstract
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.
Author(s)
시지은신하림장가영권지연김용섭
Issued Date
2021
Type
Article
Keyword
CRISPR-Casgenome engineeringbase editingBRCA1
DOI
10.3791/61557
URI
https://oak.ulsan.ac.kr/handle/2021.oak/7531
https://ulsan-primo.hosted.exlibrisgroup.com/primo-explore/fulldisplay?docid=TN_cdi_proquest_miscellaneous_2501476732&context=PC&vid=ULSAN&lang=ko_KR&search_scope=default_scope&adaptor=primo_central_multiple_fe&tab=default_tab&query=any,contains,Functional%20Assessment%20of%20BRCA1%20variants%20using%20CRISPR-Mediated%20Base%20Editors&offset=0&pcAvailability=true
Publisher
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Location
미국
Language
영어
ISSN
1940-087X
Citation Number
61557
Citation Start Page
0
Citation End Page
0
Appears in Collections:
Medicine > Medicine
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