Whole genome inducible CRISPR KO screens for HIFa activity

Hypoxic inducible factors control the cellular response low oxygen by transcription regulation of gene programs. In order to understand the genes that control HIFa transcription factor regulated gene expression we generated dual fluorescent dFLASH-HIF reporter cells lines in HEK293T and U2OS cells to monitor HIFa activity. We then generated dox-inducible Cas9-TagBFP expressing reporter lines and performed 3 replicate genome wide KO gain- or loss- of function screens for HIF activity in U2OS and HEK293T reporter cell lines using the Brunello sgRNA KO library (~79,000 sgRNA, covering ~19,000 genes). High throughput sequencing was used to identify sgRNAs in enriched vs library samples and sgRNA enrichment quantified by either DEseq2 or MAGeck. Library vs Low vs High HIF activity isolated based on DMOG induced Tomato expression in U2OS or HEK293 cell lines