Epistatic transcriptional interations underlying oncogenic genetic interactions (GI) via combinatorial CROP-seq

In our combinatorial CRISPR screenings, we screened for tumor/growth promoting GIs between 52 tumor suppressor genes (TSGs). In this part, we systematically profiled single-cell transcriptomes of double knocking-out (DKO) and single knocking-out (SKO) cells of GI pairs identified in our GI screenings, and tried to decipher the role of transcription in the phenotypic effects of GIs. Cell library expressing dual CROPseq-sgRNAs was constructed by infection of CROPseqGuide-Puro-sgRNAs viruses and CROPseqGuide-Blasti-sgRNAs viruses sequentially. MCF10A-Cas9-Venus-vector cells were infected with 15 different CROPseqGuide-Puro-sgRNA viruses individually. CROPseqGuide-puro-(NF1_1, PTEN_5, SMAD4_5, CASP8_5, CBFB_2, CDH1_5, RB1_3, TP53_3, NF2_2, TBX3_3, USP9X_3, TP53_4, TP53_5, hRosa26_2, CTRL0002), and selected in 1.5 µg/ml puromycin for 4 days. Cells were then frozen, and passed for the next infection of CROPseqGuide-Blasti viruses. After 24 h of infection, Blasticidin (15 ng/µl) was added for selection. 72 of different pair-wised sgRNA combinations in total were included in the CROPseq cell library. Cell library was then cultured in different media and harvested for single cell transcriptome profiling. Single cells contain different perturbations that generating single knocking-out (SKO) and double knocking_out (DKO) and Control cells.