Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Terkelsen T

PROVIDER: S-EPMC10870130 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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

Terkelsen Thorkild T Mikkelsen Nanna Steengaard NS Bak Ebbe Norskov EN Vad-Nielsen Johan J Blechingberg Jenny J Weiss Simone S Drue Simon Opstrup SO Andersen Henning H Andresen Brage Storstein BS Bak Rasmus O RO Jensen Uffe Birk UB

American journal of human genetics 20240124 2

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 act ...[more]

PMID: 38272032

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Project description:BackgroundPeople with chronic pain use a range of healthcare services, but they also report a high degree of dissatisfaction with treatments. One reason for dissatisfaction might be participants' expectations towards treatments. The aim of this study was to explore expectations of people with chronic pain towards participation in easily accessible pain management interventions delivered in public primary care.MethodsA qualitative study using semi-structured individual face-to-face interviews with 21 informants. The informants were recruited among participants enrolled in a randomised controlled trial on the effect of an easily accessible self-management course for people with chronic pain. The data were analysed thematically using Systematic Text Condensation.ResultsHaving experienced pain for a long time, there was no specific expectation of a cure or a significant alleviation of the pain. The informants' expectations mainly concerned a hope that participation could lead to a better everyday life. The informants said that hope was important as it motivated them to keep going and continue self-care activities. The hope acted as a driving force towards trying new interventions and maintaining motivation to do activities they experienced as beneficial. Both concrete aspects of the current intervention and an understanding of what interventions in general could offer contributed to the informants hope. The expectations centred about the interventions being something new, as they had not previously tried this service, an opportunity to gain and reinforce skills, to help them continue to grow as a person, to meet others in similar situations, and to access professional support in an easy manner. Participating in interventions provided by healthcare services was seen by some as an act of self-care, where they did something active to manage their health.ConclusionsExpectations towards the interventions were related to a hope for participation leading to a better everyday life. The role of hope for peoples' motivation to self-care implies that service providers should be aware of and help to maintain hope for a better everyday life. The importance of social support as part of self-care should be acknowledged when developing interventions targeting chronic pain.Trial registrationClinicalTrials.gov: NCT02531282 . Registered on August 21 2015.

Dataset Information

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

Publications

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

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