From GWAS signal to function: targeted CRISPR activation enables functional characterization of non-coding SNPs in...

Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with complex traits in poultry. However, most GWAS-identified variants reside in non-coding regions, making their functional relevance to their phenotypes unclear. Emerging evidence suggests that many of these markers overlap cis-regulatory elements, yet experimental validation of their biological function remains limited. Here, we investigated non-coding GWAS variants associated with nucleotide-related compounds in chicken breast muscle by targeting SNP-containing genomic regions using a CRISPR activation (CRISPRa) system in DF-1 cells and profiling transcriptomic responses via bulk RNA sequencing to assess the functional impact of activating these regions. Based on chicken muscle-specific epigenetic profiles and chromatin state annotations, we identified three significant GWAS variants on chromosome five associated with nucleotide metabolism. These variants are situated within cis-regulatory elements, specifically in intron three of DUSP8, intron one of SLC25A22, and upstream of FBXO3. To understand their functional impact, we employed an in vitro CRISPRa system with targeted guide RNAs to activate each non-coding SNP region in DF-1 cells. This activation resulted in significant changes at the transcriptomic level. Subsequent functional enrichment analysis of the differentially expressed genes consistently highlighted muscle-related pathways across all SNPs, including MAPK signaling, cytoskeletal remodeling, and ECM-receptor interactions, which are potentially involved in regulating nucleotide metabolism and deposition in muscle. Furthermore, transcript-level analysis of RNA-seq reads revealed that the non-coding SNP region within the intron three of DUSP8 may function as an alternative promoter, resulting in significantly higher expression of a shorter transcript that could generate a non-canonical protein isoform. Our study demonstrates that activating genomic regions harboring specific non-coding GWAS SNPs can modulate gene expression, suggesting that these SNPs may contribute to gene regulatory functions. Importantly, this work underscores the powerful utility of CRISPRa as a functional genomics tool for linking GWAS signals to their biological roles in chickens by targeting SNP-containing regions and uncovering consequential molecular phenotypes.