全基因组关联研究以及表达定量性状位点（eQTL）映射已经在前列腺癌（PCa）中鉴定出数百个单核苷酸多态性（SNP）及其靶标基因，然而对这些风险位点的功能特征的表征仍然具有挑战性。为了筛选潜在的调控SNP，我们设计了一个CRISPRi文库，其中包含9,133个指导RNA（gRNAs），以覆盖2,166个与PCa相关的候选SNP位点，并确定出117个SNPs可以调控90个基因促进PCa细胞生长优势。其中，rs60464856被多个显著耗竭筛选的gRNAs覆盖（FDR < 0.05）。在PRACTICAL和FinnGen队列的合并SNP关联分析中显示rs60464856 G等位基因的PCa风险显著增加（p值= 1.2 × 10-16和3.2 × 10-7，分别）。随后的eQTL分析显示G等位基因与多个数据集中RUVBL1表达的增加相关。进一步的CRISPRi和xCas9碱基编辑实验证实rs60464856 G等位基因导致RUVBL1表达上调。此外，SILAC基于蛋白质组学的分析显示在rs60464856区域存在着凝聚素亚单位的等位结合，而HiC数据集显示在前列腺细胞系中存在一致的染色质相互作用。RUVBL1的降低抑制了PCa细胞的增殖以及在异种移植小鼠模型中的肿瘤生长。基因集富集分析表明RUVBL1表达与细胞周期相关通路有关。RUVBL1的表达增加以及细胞周期通路的激活与TCGA队列中的PCa生存率下降相关。我们的CRISPRi筛选重点研究了约一百个对前列腺细胞增殖至关重要的调控SNPs。结合蛋白质组学和功能研究，我们揭示了rs60464856和RUVBL1在PCa进展中的机制作用。© 2023美国人类遗传学学会。由Elsevier Inc.出版。版权所有。

Genome-wide association studies along with expression quantitative trait locus (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 9,133 guide RNAs (gRNAs) to cover 2,166 candidate SNP loci 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 screening (FDR < 0.05). Pooled SNP association analysis in the PRACTICAL and FinnGen cohorts showed significantly higher PCa risk for the rs60464856 G allele (p value = 1.2 × 10-16 and 3.2 × 10-7, respectively). Subsequent eQTL analysis revealed that the G allele is associated with increased RUVBL1 expression in multiple datasets. Further CRISPRi and xCas9 base editing confirmed that the rs60464856 G allele leads to elevated RUVBL1 expression. Furthermore, SILAC-based proteomic analysis demonstrated allelic binding of cohesin subunits at the rs60464856 region, where the HiC dataset showed consistent chromatin interactions in prostate cell lines. RUVBL1 depletion inhibited PCa cell proliferation and tumor growth in a xenograft mouse model. Gene-set enrichment analysis suggested an association of RUVBL1 expression with cell-cycle-related pathways. Increased expression of RUVBL1 and activation of cell-cycle pathways were correlated with poor PCa survival in TCGA datasets. 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 RUVBL1 in PCa progression.Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.