Lentiviral particles containing pMH0001 (UCOE-SFFV-dCas9-BFP-KRAB, Addgene #85969) were produced by transfecting HEK293T cells with standard packaging vectors using the TransIT-Lenti Transfection Reagent (Mirus Bio, MIR 6605). M10G cells were stably transduced with lentiviral particles to generate M10GdCas9-KRAB cells by isolating BFP-expressing cells using fluorescence activated cell sorting on a Sony SH800.
Single-guide RNA (sgRNA) protospacer sequences were individually cloned into the pCRISPRia-v2 vector (Addgene plasmid #84832), between the BstXI and BlpI sites, by ligation. Each vector was verified by Sanger sequencing of the protospacer. One sgRNA expression vector with the following protospacer was cloned for non-targeting control sgRNAs (ncRNA) (5′-GCTGCATGGGGCGCGAATCA-3′), PTCH1 sgRNAs (sgPTCH1) (5′-AAATGTACGAGCACTTCAAG-3′) and SMO sgRNAs (sgSMO) (5′-CAAGAACTACCGATACCGTG-3′). Lentivirus was generated as described above for each sgRNA expression vector, and M10GdCas9-KRAB cells were independently transduced with lentivirus from each sgRNA expression vector, then selected to purity using 20 μg/mL puromycin over 7 days.
Single-guide RNA (sgRNA) protospacer sequences were individually cloned into the pCRISPRia-v2 vector (Addgene plasmid #84832), between the BstXI and BlpI sites, by ligation. Each vector was verified by Sanger sequencing of the protospacer. One sgRNA expression vector with the following protospacer was cloned for non-targeting control sgRNAs (ncRNA) (5′-GCTGCATGGGGCGCGAATCA-3′), PTCH1 sgRNAs (sgPTCH1) (5′-AAATGTACGAGCACTTCAAG-3′) and SMO sgRNAs (sgSMO) (5′-CAAGAACTACCGATACCGTG-3′). Lentivirus was generated as described above for each sgRNA expression vector, and M10GdCas9-KRAB cells were independently transduced with lentivirus from each sgRNA expression vector, then selected to purity using 20 μg/mL puromycin over 7 days.