Genetic landscape and functional exploration of kidney cancer predisposition causality in cross-ancestral populations [screen]

To functionally explore renal cell carcinoma (RCC) susceptibility variants, we performed CUT&Tag for H3K27ac and ATAC-seq on 769P cells to assess transcriptional activity. Subsequently, we conducted a CRISPR screen and CRISPR droplet sequencing (CROP-seq), which combines pooled CRISPR screens with single-cell RNA sequencing (scRNA-seq), to identify target genes potentially regulated by likely causal SNPs. Functionally, we established a novel association between rs28684409 and the oncogene RPL4 at the complex genetic locus 15q22.31. As the next step, we performed CRISPRi on rs28684409 and conducted RNA-seq to identify differential gene expression associated with this variant. Overall design: CRISPRi/a (786-O and Caki-1) cells were transduced with the pooled sgRNA library at low MOI of 0.5.Twenty-four hours after infection, cells were selected using puromycin (2 µg/ml) for 7 days. Cells were split into triplicates every 3 days, and maintained at 300X coverage throughout the screen. Cells were harvested in replicates on day 0, day 14 and day 21 after puromycin selection. sgRNA was amplified by PCR using specific primers, the amplified products were sent to the Beijing Genomics Institute (BGI) for library construction and sequencing.