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

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 CROP-seq library at high MOI. Cells were selected using puromycin (2 ug/ml) for 3 days after transduction, after which they were expanded and harvested at day 7. The cells were further processed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) (10x Genomics) according to the manufacturer's protocol. To enrich for sgRNA transcripts, PCR was performed on 10 ng of cDNA from the 3' single-cell RNA libraries using specific primers. The libraries were sequenced on Illumina (Novogene), using a configuration identical to that of standard 10x Genomics libraries.