From GWAS Signal to Function: Targeted CRISPR Activation Enables Functional Characterization of Non-Coding SNPs in Chickens

Jaewon Kim¹Jeong Hoon²Minjun Kim¹Grace Schmidt²Eunjin Cho¹Jun Heon Lee^1*Tae Hyun Kim^2*

• ¹Chungnam National University, Yuseong-gu, Republic of Korea
• ²The Pennsylvania State University (PSU), University Park, United States

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 5 associated with nucleotide metabolism. These variants are situated within cis-regulatory elements, specifically in intron 3 of DUSP8, intron 1 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 3 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.