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

Area of Science:

• Genetics
• Molecular Biology
• Diagnostic Development

Background:

• Genetic variants impacting mRNA splicing cause hereditary disorders.
• Predicting variant spliceogenicity is difficult.
• Current RNA diagnostics are limited to genes expressed in accessible tissues.

Purpose of the Study:

• To develop a novel ex vivo splicing assay using CRISPR activation (CRISPRa).
• To enable functional characterization of splice-altering variants in genes not expressed in accessible tissues.
• To establish a new diagnostic tool for monogenic disorders.

Main Methods:

• CRISPR activation (CRISPRa) via a dCas9-VPR mRNA delivery platform.
• Induction of gene expression in skin fibroblasts.
• Splice variant profiling using RT-PCR, next-generation sequencing, and long-read sequencing.
• Characterization of variants in myelin protein zero (MPZ) and spastin (SPAST) genes.

Main Results:

• CRISPRa successfully upregulated MPZ and SPAST transcription in skin fibroblasts.
• The assay enabled detailed splice variant profiling.
• Demonstrated proof of principle for CRISPRa-based genetic diagnostics.

Conclusions:

• CRISPRa is a promising tool for activating gene expression in accessible cells for variant impact studies.
• This method overcomes limitations of current RNA diagnostics for inaccessible gene tissues.
• The procedure is practical for diagnostic laboratories.