Figure 3
Schematic of the CRISPR library construction and HCV screening.
(a) The structure of the lentiviral plasmid expressing OCT1 and Cas9. (b) Indels induced by the lentivirus-delivered sgRNA (5-TTGGCCAGACTTGCATCCG-3) targeting the CSPG4 gene in the indicated cells were assayed by T7E1 digestion. Genomic DNA from HeLaOC-SC31 was used as a positive control and the wild type (WT) Huh7.5(NIrD) was used as a negative control. (c) Schematic of the sgRNA library screening. sgRNAs were delivered into Huh7.5(NIrD)OC-SC cells by lentiviral infection with a MOI of 0.1. Three replicates of the libraries were challenged with 3–4 rounds of HCVcc, followed by FACS sorting to enrich the mCherry-negative clones. A comparison of the abundance of sgRNAs between the treated and untreated populations through high-throughput sequencing analysis was conducted following the same procedure as previously reported31.
