Abstract
Most genetic loci linked to polygenic traits are in non-coding regions, with complex regulation and linkage disequilibrium (LD), complicating causal variant and gene prioritization. We used multiplexed single-cell CRISPR interference and activation perturbations to investigate cis-regulatory element (CRE) and gene expression relationships within tight LD in the endogenous chromatin context. We demonstrated the prevalence of multiple causality in perfect LD (pLD) for independent expression quantitative trait loci (eQTLs) and uncovered fine-grained genetic effects on gene expression within pLD, which are difficult to decipher using traditional eQTL fine-mapping or existing computational methods. We found that over one-third of the causal CREs lack classical epigenetic markers prior to perturbation, and we functionally validated one of these hidden regulatory mechanisms. Leveraging Multiome single-cell epigenetic and sequence perturbations, we highlighted the regulatory plasticity of the human genome. Our study will guide the exploration of missing causal mechanisms underlying molecular trait regulation and disease development.