CRISPR activation to characterize splice-altering variants in easily accessible cells.

Autor:	Terkelsen T; Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark; Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address: thorkild.terkelsen@clin.au.dk., Mikkelsen NS; Department of Biomedicine, Aarhus University, Aarhus, Denmark., Bak EN; Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark., Vad-Nielsen J; Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark., Blechingberg J; Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark., Weiss S; Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark., Drue SO; Department of Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark., Andersen H; Department of Neurology, Aarhus University Hospital, Aarhus, Denmark., Andresen BS; Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark., Bak RO; Department of Biomedicine, Aarhus University, Aarhus, Denmark., Jensen UB; Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark; Department of Biomedicine, Aarhus University, Aarhus, Denmark. Electronic address: uffejens@rm.dk.
Jazyk:	angličtina
Zdroj:	American journal of human genetics [Am J Hum Genet] 2024 Feb 01; Vol. 111 (2), pp. 309-322. Date of Electronic Publication: 2024 Jan 24.
DOI:	10.1016/j.ajhg.2023.12.024
Abstrakt:	Genetic variants that affect mRNA splicing are a major cause of hereditary disorders, but the spliceogenicity of variants is challenging to predict. RNA diagnostics of clinically accessible tissues enable rapid functional characterization of splice-altering variants within their natural genetic context. However, this analysis cannot be offered to all individuals as one in five human disease genes are not expressed in easily accessible cell types. To overcome this problem, we have used CRISPR activation (CRISPRa) based on a dCas9-VPR mRNA-based delivery platform to induce expression of the gene of interest in skin fibroblasts from individuals with suspected monogenic disorders. Using this ex vivo splicing assay, we characterized the splicing patterns associated with germline variants in the myelin protein zero gene (MPZ), which is exclusively expressed in Schwann cells of the peripheral nerves, and the spastin gene (SPAST), which is predominantly expressed in the central nervous system. After overnight incubation, CRISPRa strongly upregulated MPZ and SPAST transcription in skin fibroblasts, which enabled splice variant profiling using reverse transcription polymerase chain reaction, next-generation sequencing, and long-read sequencing. Our investigations show proof of principle of a promising genetic diagnostic tool that involves CRISPRa to activate gene expression in easily accessible cells to study the functional impact of genetic variants. The procedure is easy to perform in a diagnostic laboratory with equipment and reagents all readily available. Competing Interests: Declaration of interests R.O.B. holds equity in Graphite Bio and UNIKUM Tx and is a paid consultant of UNIKUM Therapeutics. (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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