Abstract 446: CRISPRi-SNPs-seq identified regulatory loci conferring prostate cancer

Abstract

Introduction: Genome-wide association studies (GWAS) along with expression quantitative trait loci (eQTL) have identified hundreds of genetic variants and target genes in prostate cancer (prCa). Although genetic predisposition has mainly been described in prostate cancer (PrCa), functional characterization of these risk loci remains a challenge. Methods: Low multiplicity of infection creates single lentiviral integrated cell population, which enable us to evaluate biological significance of steric hindrance at certain SNP sites in large scale. To screen for regulatory SNP, we designed a guide RNA library to target 2166 potential functional SNP sites with CRISPOR software. We performed negative screening in dCas9-KRAB stable prostate cell lines and applied RIGOR program to discover the SNPs that are essential for cell proliferation. We further validated regulatory role of selected SNPs using luciferase reporter assay, ChIP-qPCR and CRISPR-based SNP editing in prostate cells. Results: After gRNA interfering for 21 days, we performed RIGOR analysis and identified 153 proliferation-essential SNPs, covered by one or multiple prostate cancer cell lines. Intersection analysis showed that these SNPs tended to reside in 5'-UTR and intron regions. To characterize regulatory role of these SNPs, we performed functional analysis in a SNP rs60 since prostate cells containing guide RNAs targeting rs60 were significantly depleted (FDR<0.05). Pooled GWAS SNP analysis showed that the G allele of this SNP exhibit 10% higher PrCa risk than A allele. eQTL analysis showed that the G allele is associated with an increased expression of RUVBL1 (FDR=3.3E-13). To further characterize the SNP-gene association, we performed CRISPR-based RNA interference analysis that targeted the SNP site. We observed significant inhibition of the RUVBL1 gene expression in prostate cell lines. We also applied xCas9 adenine base editor and successfully converted rs60 A into G allele in multiple clones of BPH1, DU145 and PC3 cell line. Consequently, we observed RUVBL1 expression increase after switching A to G allele at this locus. We performed ChIP-qPCR and confirmed rs60 flanking region was highly H3K4me3 modified, suggesting a transactivation role of this loci in regulating RUVBL1 expression. When stably knocked down RUVBL1 expression by shRNA we found significant proliferation inhibition in multiple prostate cell lines. Gene set enrichment analysis showed an enrichment of MYC target genes with RUVBL1 expression in TCGA prostate cancer cohorts. Furthermore, increased RUVBL1 expression was associated with poor progression-free survival (p<0.01) in TCGA datasets. Conclusion: CRISPRi-SNPs-seq is a powerful screening tool to identify regulatory SNPs essential for cell proliferation. In combination with in-depth functional assays, the technology will facilitate discovery of regulatory variants and their genes responsible for disease risk. Citation Format: Yijun Tian, Jong A. Park, Liang Wang. CRISPRi-SNPs-seq identified regulatory loci conferring prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 446.