Title: Characterizing human regulatory genetic variation using CRISPR/Cas9 genome editing
Description: Rare gene-disrupting variants and common regulatory variants play key roles in rare and common disease, respectively. These variants are of great interest for investigation into genetic contributions to disease, but experimental methods to validate their impact on gene expression levels are lacking. In this study, we utilized CRISPR/Cas9 genome editing to validate regulatory variants including cis-eQTLs, rare stop-gained variants in healthy and disease cases and one immune-response trans-eQTL master regulator. For investigation into common and rare regulatory variants within transcribed regions, we developed a scalable CRISPR-based polyclonal assay for experimental assessment. First, we applied this assay to nine rare stop-gained variants found in the general population, in GTEx. After editing, the stop-gained variants show a significant allele-specific depletion in transcript abundance, as expected. Next, we utilized the assay to validate 33 common eQTLs found in GTEx. After editing, the eQTL variants show higher variance in effect size than control variants, indicating a regulatory effect.

Finally, we applied the polyclonal editing approach to clinical and new stop-gained variants in two disease-associated genes. The results follow the expected trend, with NMD being triggered by variants upstream of the NMD threshold but not by those beyond. This method demonstrates scalable experimental confirmation of putative causal regulatory variants, and improved interpretation of regulatory variation in humans. Next, we sought to experimentally validate an immune-response eQTL for IRF1 in cis and many genes in trans under LPS stimulation. We used CRISPRi to repress the enhancer locus and found that the enhancer is active in our immune cell system. Next, we used CRISPR-Cas9 genome editing and isolation of monoclonal cell lines to target this variant locus. After LPS stimulation, we performed RNA-sequencing on wild type and edited clones, showing that the effect size of the genes which are associated with the trans-eQTL are correlated with differential expression between the edited and wild type cell lines for the same genes.

Additionally, we find that the differential expression between edited clones is correlated with CRISPRi repression of the IRF1 promoter and enhancer. In this way, we were able to identify a common genetic variant which modifies the transcriptomic immune response to LPS and validate the trans-eQTL signal.