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.
Subject(s)
CRISPR-Cas Systems , Gene Editing , CRISPR-Cas Systems/genetics , Gene Editing/methods , Cytosine , Nucleotides , Genes, Tumor SuppressorABSTRACT
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.
Subject(s)
BRCA1 Protein/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing , Genetic Variation , Base Sequence , Breast Neoplasms/genetics , CRISPR-Associated Protein 9/metabolism , Cell Line, Tumor , Female , High-Throughput Nucleotide Sequencing , Humans , RNA, Guide, Kinetoplastida/geneticsABSTRACT
CRISPR-based base editors (BEs) are widely used to induce nucleotide substitutions in living cells and organisms without causing the damaging DNA double-strand breaks and DNA donor templates. Cytosine BEs that induce C:G to T:A conversion and adenine BEs that induce A:T to G:C conversion have been developed. Various attempts have been made to increase the efficiency of both BEs; however, their activities need to be improved for further applications. Here, we describe a fluorescent reporter-based drug screening platform to identify novel chemicals with the goal of improving adenine base editing efficiency. The reporter system revealed that histone deacetylase inhibitors, particularly romidepsin, enhanced base editing efficiencies by up to 4.9-fold by increasing the expression levels of proteins and target accessibility. The results support the use of romidepsin as a viable option to improve base editing efficiency in biomedical research and therapeutic genome engineering.
Subject(s)
Adenine , CRISPR-Cas Systems , Gene Editing , Histone Deacetylase Inhibitors/pharmacology , Depsipeptides/pharmacology , Doxycycline/pharmacology , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Luminescent Agents/analysis , Protein Biosynthesis , RNA/biosynthesisABSTRACT
Although prime editors are a powerful tool for genome editing, which can generate various types of mutations such as nucleotide substitutions, insertions, and deletions in the genome without double-strand breaks or donor DNA, the conventional prime editors are still limited to their target scopes because of the PAM preference of the Streptococcus pyogenes Cas9 (spCas9) protein. Here, we describe the engineered prime editors to expand the range of their target sites using various PAM-flexible Cas9 variants. Using the engineered prime editors, we could successfully generate more than 50 types of mutations with up to 51.7% prime-editing activity in HEK293T cells. In addition, we successfully introduced the BRAF V600E mutation, which could not be induced by conventional prime editors. These variants of prime editors will broaden the applicability of CRISPR-based prime editing technologies in biological research.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Genetic Engineering , Nucleotide Motifs , Alleles , Amino Acid Substitution , Binding Sites , CRISPR-Associated Protein 9 , Clustered Regularly Interspaced Short Palindromic Repeats , Genetic Engineering/methods , HEK293 Cells , Humans , Mutation , Proto-Oncogene Proteins B-raf/geneticsABSTRACT
Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.
