CRISPR Inhibition of KSHV Transcripts

sgRNAs for CRISPRi of KSHV (accession number GQ994935.1) were designed as described by Horlbeck et al. [33 (link)] (Table S1). Synthetic DNA segments encoding the sgRNAs were cloned into the pHR-SFFV-dCas9-BFP-KRAB (Addgene 46911) at BstXI and XhoI, and the clones were confirmed by sanger sequencing. Lentiviral production and transduction were performed as described above. After transduction, BCBL-1 cells were maintained in 1 μg/mL of puromycin for 10 days prior to the selection of BFP+/sgRNA+ by FACS in a Sony SH800 instrument. iSLK-219 cells were selected for BFP+/sgRNA+ expression by FACS in a Sony SH800 instrument (see Tables S1 and S2 for sgRNA sequences and genomic coordinates).