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. 2015 Jun 2;5:10833. doi: 10.1038/srep10833

Figure 2. Sustained expression of CRISPR machinery enables large reductions in HBV DNA and cccDNA.

Figure 2

(a) Schematic of lentiviral vector and experimental strategy for sustained CRISPR expression. (b-c) CRISPR constructs targeting HBV cause progressive reduction in (b) cccDNA and (c) total HBV DNA levels dependent on successful targeting of viral DNA and Cas9 nuclease activity; data shown are from one representative experiment pooled across 3 separate HBV-targeting guides (sg6, sg17, sg21), and consistent across multiple independent transduction experiments. (d) Southern blot of HBV DNA forms using Hirt’s extraction (to deplete high-molecular weight DNA), shows HBV-targeted sgRNAs with nuclease-active Cas9 generate near-total reduction in cccDNA. (e) Surveyor assay to detect indel formation in total HBV DNA (top) and episomal HBV DNA, enriched by treatment with plasmid-safe DNase (bottom); lentiviral transduction enables high levels of cutting of HBV. Arrowheads depict surveyor digestion products. Expected PCR product sizes for sg6, sg17 and sg21 are respectively 599, 946 and 507 bp. Approximate sizes of surveyor digestion products for sg6, sg17 and sg21 are respectively: 429 + 170, 570 + 376, 275 + 232. (f) Immunofluorescent imaging of HBV Core protein demonstrates large reduction in Core staining upon targeting by sg17 specifically against the Core ORF. (g-h) Cas9/gRNA-transduced Hep-NTCP cells are cocultured with HepG2.2.15 cells to infect them with HBV followed by depletion of HepG2.2.15 cells using puromycin selection (Schematic in Fig S6 left). (g) From left to right, HBsAg secretion, cccDNA copies, levels of HBV 3.5kb RNA relative to 5 bp mismatch control, and titer of HBV DNA in culture medium show that Cas9/sg17 reduce HBV infection in de novo infection. 17M: 5 bp mismatch control. 17D: dead Cas9 with g17. Data shown are from one representative experiment, and consistent across experiments. (h) Surveyor assay performed on DNA untreated (left) or treated (right) with Plasmid-Safe DNase to remove non-episomal viral forms. Arrowheads indicate indel formation. (b-c) *p < 0.05 for selected comparison; **p < 0.01 for selected comparison, as assessed by one-way ANOVA with Tukey post-hoc test.