Combined CRISPRi and proteomics screening reveal a cohesin-CTCF-bound allele contributing to increased expression of and prostate cancer progression.

Genome-wide association studies along with expression quantitative trait loci (eQTL) mapping have identified hundreds of single nucleotide polymorphisms (SNPs) and their target genes in prostate cancer (PCa), yet functional characterization of these risk loci remains challenging. To screen for potential regulatory SNPs, we designed a CRISPRi library containing 9133 guide RNAs (gRNAs) to target 2,166 candidate SNP sites implicated in PCa and identified 117 SNPs that could regulate 90 genes for PCa cell growth advantage. Among these, rs60464856 was covered by multiple gRNAs significantly depleted in the screening (FDR<0.05). Pooled SNP association analysis in the PRACTICAL and FinnGen cohorts showed significantly higher PCa risk for the rs60464856 G allele (pvalue=1.2E-16 and 3.2E-7). Subsequent eQTL analysis revealed that the G allele is associated with increased expression in multiple datasets. Further CRISPRi and xCas9 base editing proved the rs60464856 G allele leading to an elevated expression. Furthermore, SILAC-based proteomic analysis demonstrated allelic binding of cohesin subunits at the rs60464856 region, where HiC dataset showed consistent chromatin interactions in prostate cell lines. depletion inhibited PCa cell proliferation and tumor growth in xenograft mouse model. Gene set enrichment analysis suggested an association of expression with cell-cycle-related pathways. An increased expression of and activations of cell-cycle pathways were correlated with poor PCa survival in TCGA datasets. Together, our CRISPRi screening prioritized about one hundred regulatory SNPs essential for prostate cell proliferation. In combination with proteomics and functional studies, we characterized the mechanistic role of rs60464856 and in PCa progression.