functional-assessment-brca1-variants-using-crispr-mediated-base

Functional Assessment of <em>BRCA1</em> 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 ...

University of Ulsan College of Medicine

The bacteria-derived clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are powerful tools for genome engineering. Recently, in addition to Cas protein engineering, the improvement of guide RNAs are also performed, contributing to broadening the research area of CRISPR-Cas9 systems. Here we develop a fusion guide RNA (fgRNA) that functions with both Cas9 and Cpf1 proteins to induce mutations in human cells. Furthermore, we demonstrate that fgRNAs can be used in multiplex genome editing and orthogonal genome manipulation with two types of Cas proteins. Our results show that fgRNAs can be used as a tool for performing multiple gene manipulations.

Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

The originally published version of this Article contained an error in the spelling of the author Da-eun Kim, which was incorrectly given as Da-Eun Kim. Furthermore, in Figure 1a, the Cas9 protein was positioned incorrectly during typesetting. These errors have now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1.

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.

A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.

Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.

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.

Article	Year
Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature communications\| PubMed ID: 29167440	2017
Publisher Correction: Fusion guide RNAs for orthogonal gene manipulation with Cas9 and Cpf1. Nature communications\| PubMed ID: 29335559	2018
A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants. Oncogene\| PubMed ID: 31467430	2020
Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32926383	2021
Small-molecule inhibitors of histone deacetylase improve CRISPR-based adenine base editing. Nucleic acids research\| PubMed ID: 33544854	2021

Ha Rim Shin

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