CRISPR/Cas9-mediated gene knockout

We used the CRISPR/Cas9 system to generate gene-specific knockout cells. We designed single guided RNAs (sgRNAs) using a public domain web-based CRISPR sgRNA design tool CHOPCHOP (https://chopchop.cbu.uib.no). Table S1 lists individual sgRNA sequences targeting different genes. When generating Human VHL-KO, human STING-KO, human HIF1α-KO, human HIF2α-KO, mouse Sting-KO, and mouse Mda5-KO human and murine cancer cells, we used the lentiCRISPRv2 vector (Addgene #52961) following a published protocol from the Zhang lab.⁸² We digested LentiCRISPRv2 with BsmBI (NEB; Cat #R0580) and gel purified it using the Gene JET Gel extraction kit (Thermo Fisher Scientific; Cat #K0692). Oligos encoding sgRNA sequences were phosphorylated, annealed, and subsequently ligated into digested LentiCRISPRv2.

To produce sgRNA-encoding lentivirus vectors, we used HEK293T cells co-transfected with lentiviral constructs encoding the target sgRNAs and second-generation packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) following the instruction from the Trono Lab (https://www.epfl.ch/labs/tronolab/laboratory-of-virology-and-genetics/lentivectors-toolbox/).

To generate clonal knockout cell lines, we infected target cells with sgRNA-encoding CRISPR/Cas9 lentivirus and cultured them in a complete cell growth medium, selected with puromycin (1 μg/ml for Caki-1) for 7–10 days. Cells were then collected to test the expression of target genes by immunoblot.

To generate clonal VHL-KO Caki-1 cells, cells were seeded in 96-well plates after a 7-day puromycin selection and screened for pure knockout clones verified by immunoblot analysis. To generate VHL/STING double knockout (DKO) cells, including VHL/HIF1α DKO, VHL/HIF2α DKO, or VHL/HIF1α/HIF2α triple knockout (TKO) Caki-1 cells, we cloned sgRNAs sequences encoding HIF1α, and/or HIF2α into digested lentiCRISPRv2 neo vector (Addgene #98292), respectively. We then infected VHL-KO Caki-1 cells with the sgRNA-encoding lentivirus and selected the cells with neomycin (2 mg/ml) for 10–14 days. Lentivirus prepared from lentiCRISPRv2 neo vector was used to generate control cells. We then detected protein levels of STING, HIF1α, and/or HIF2α knockdown (KD) by western blot. To generate VHL/STING DKO and VHL/MDA5 DKO Caki-1 and MC38 cells, we infected VHL-KO Caki-1 cells with sgRNA-encoding lentivirus and cultured them in complete DMEM medium, and selected with puromycin (5 μg/ml for MC38 cells and 1 μg/ml for Caki-1 cells) for 7 days. We then seeded the cells in 96-well plates for single clone selection. We then verified the cells’ VHL/STING DKO and VHL/MDA5 DKO status by immunoblot analysis.

To generate Vhl knockout in Renca, B16, or MC38 cells, we digested the px330-mcherry (Addgene #98750) and pSpCas9(BB)-2A-GFP(PX458) (Addgene #48138) with BbsI (NEB; Cat #R0539S), and then gel-purified, and ligated the vectors with annealed oligos encoding sgRNA_1 or sgRNA_2 targeting Vhl. We then transfected the vectors in the murine tumor cells with sgRNA-encoding constructs using Lipofectamine 2000 (Thermo Fisher Scientific; Cat #11668019). Cells transiently transfected with px330-mCherry and PX458 vectors alone were as controls (VC). We then cultured the cells using a complete DMEM medium [DMEM with 10% FBS and 100U/ml penicillin-Streptomycin (Gibco by Life Technologies; Cat #15140122)] for 6 days and subjected them to FACS sorting (Duke Cancer Institute flow cytometry core facility). We then seeded GFP⁺ mCherry⁺ cells in 96 well plates for single clone selection. We screened for VHL-KO single clones by immunoblot analysis. Table S1 shows the sgRNA primer sequences used for targeting various genes in this study.