A polyclonal allelic expression assay for detecting regulatory effects of transcript variants
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A polyclonal allelic expression assay for detecting regulatory effects of transcript variants Margot Brandt1,2, Alper Gokden1, Marcello Ziosi1 and Tuuli Lappalainen1,2*
Abstract We present an assay to experimentally test the regulatory effects of genetic variants within transcripts using CRISPR/ Cas9 followed by targeted sequencing. We applied the assay to 32 premature stop-gained variants across the genome and in two Mendelian disease genes, 33 putative causal variants of eQTLs, and 62 control variants in HEK293T cells, replicating a subset of variants in HeLa cells. We detected significant effects in the expected direction (in 60% of variants), demonstrating the ability of the assay to capture regulatory effects of eQTL variants and nonsense-mediated decay triggered by premature stop-gained variants. The results suggest a utility for validating transcript-level effects of genetic variants. Keywords: CRISPR/Cas9 genome editing, Variant validation, Regulatory variation, eQTL, Nonsense-mediated decay
Background The interpretation of the functional effects of common and rare variants in the human population is a major objective in human genetics and genomics. Despite the success of mapping genetic associations to complex traits by genomewide association studies (GWAS) and interpreting their effects on gene expression by expression quantitative trait loci (eQTL) studies [1–5], the causal variants at GWAS loci and eQTLs are usually unknown due to linkage disequilibrium (LD). Statistical fine-mapping methods [6–8] can help narrow down causal variants, but experimental validation of the performance of these methods is lacking. For rare variants, functional interpretation has distinct challenges even in the well-annotated coding regions. Rare disease studies often result in hundreds to thousands of potential disease-causing variants identified from whole-exome sequencing, and prioritization based on their functional effect is essential for research and clinical use [9].
* Correspondence: [email protected] 1 New York Genome Center, New York, NY, USA 2 Department of Systems Biology, Columbia University, New York, NY, USA
Thus, there is a need for experimental methods to confirm the effects of common and rare variants. Methods such as massively parallel reporter assays (MPRAs) [10, 11], which couple regulatory sequences with an expression-correlated reporter, are highthroughput approaches for finding active regulatory variants outside of the gene body, and analogous methods exist for variants affecting splicing [12, 13]. However, the results of the assays show low concordance with eQTL data [10, 11], perhaps due to taking the variant out of its genomic context. Furthermore, MPRAs are not suited to testing variants within the transcript that can affect gene expression levels via post-transcriptional mechanisms, e.g., RNA stability. Regulatory variants are strongly enriched not only in promoters and enhancers, but also in UTRs and other transcript annotations [1], emphasizing the need for a met
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