A polyclonal allelic expression assay for detecting regulatory effects of transcript variants

Area of Science:

• Genetics
• Molecular Biology
• Bioinformatics

Background:

• Genetic variants can alter gene regulation and cause disease.
• Existing methods may not fully capture transcript-level regulatory effects.
• Premature stop-gained variants can trigger nonsense-mediated decay.

Purpose of the Study:

• To develop and validate a novel assay for experimentally assessing the regulatory effects of genetic variants on transcripts.
• To evaluate the assay's ability to detect effects of premature stop-gained variants and expression quantitative trait loci (eQTL) variants.
• To demonstrate the utility of the assay for validating transcript-level variant impacts.

Main Methods:

• CRISPR/Cas9 gene editing was employed to introduce specific genetic variants into cellular transcripts.
• Targeted sequencing was used to quantify the effects of these variants.
• The assay was applied to premature stop-gained variants, eQTL variants, and control variants in HEK293T and HeLa cells.

Main Results:

• The assay detected significant regulatory effects in 60% of tested variants in the expected direction.
• The assay successfully captured the regulatory impact of eQTL variants.
• The assay identified nonsense-mediated decay triggered by premature stop-gained variants.

Conclusions:

• The developed CRISPR/Cas9-based assay is effective for experimentally testing the regulatory effects of genetic variants at the transcript level.
• The assay demonstrates utility in validating the functional consequences of genetic variants, including those associated with Mendelian diseases and eQTLs.
• This approach provides a valuable tool for understanding genetic variant function and its role in human health and disease.