Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus

Abstract

Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.

Keywords: Antiviral; Cure; Liver; Mutations; NHEJ; Therapeutics.

Fig. 1

Anti-HBV activity of orthologous CRISPR/Cas9 type II systems. a–d Conservation of orthologous CRISPR/Cas9 targets across HBV genotypes A–H and anti-HBV activity of various gRNAs as determined by measuring pgRNA and S-RNA levels. pgRNA and S-RNA levels were normalized to Cas9 mRNA expression. Conservation of a specific locus was analyzed by directly mapping gRNA sequences in HBV genomes of different genotypes (A–H) (left side of the graphs). Intensities of red and green colors indicate low and high rates of conservation, correspondingly. ⁺p < 0.05, ^#p < 0.01, ^~p < 0.001, *p < 0.0001

Fig. 2

Inhibition of HBV protein production. HBsAg levels in culture medium after transfection of a SpCas9 and b StCas9 systems. c Reduction of HBcAg expression by various CRISPR/Cas9 systems 5 days post-transfection. HepG2 cells transfected with HBV-expressing plasmid and the indicated CRISPR/Cas9 system were co-stained for HBcAg (green) and SpCas9 (red) protein. Cell nuclei were labeled by Hoechst33342 dye (blue). Quantitative analysis of HBcAg-positive and SpCas9-positive cells after transfection of d SpCas9 or e StCas9 CRISPR/Cas9 systems from an experiment as described in c. NT not transfected

Fig. 3

Antiviral activity of orthologous CRISPR/Cas9 and effects of rcccDNA methylation on anti-HBV activity. Anti-HBV effects of orthologous CRISPR/Cas9 (Nm, Fn, St and Sp) were analyzed by rcccDNA transfection. Antiviral activity of NmCas9 and FnCas9 in a HepG2 transfection model were analyzed by measuring pgRNA and S-RNA (a, c) normalized to NmCas9 and FnCas9 mRNA, and HBV DNA and cccDNA levels (b, d) relative to β-globin. e–l Comparison of HBV intermediates in HepG2 cells after transfection of unmethylated or methylated rcccDNA. Effects of St and Sp CRISPR/Cas9 on e, g, i, k pgRNA and S-RNA levels (relative to Cas9 mRNA levels), f, h, j, l HBV DNA and rcccDNA (relative to β-globin) in unmethylated and methylated rcccDNA. Asterisks indicate statistically significant differences. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Deep sequencing of on-target sites in methylated and unmethylated cccDNA. Detection of indel mutations in unmethylated or methylated rcccDNA by St CRISPR/Cas9 a St3, b St4 and c St10, shown as number of mutations per 1000 reads. X-axis indicates the target regions in HBV genome, PAM protospacer adjacent motif

Fig. 5

CRISPR/Cas9-mediated suppression of HBV replication in stable cell lines. Experimental design in a HepG2-1.1merHBV and f HepG2-1.5merHBV cell lines. Suppression of HBV transcription by b, c Sp and g, h St CRISPR/Cas9. Alterations in HBV DNA and cccDNA levels (relative to β-globin) post d, e SpCas9 and i, j StCas9 transfection. k Inhibition of HBV cccDNA degradation by NU7026. Cells were mock-transfected or transfected with CRISPR/Cas9 and Sp20 gRNA (Sp20) and treated by NU7026 for 3 days or DMSO as control. l Frequency of indel mutations in mock and Sp20 treated by DMSO or NU7026. Distribution of m insertions and n deletions by length in experimental groups. Numbers indicate the size intervals of indels. o The proposed model of HBV cccDNA degradation by CRISPR/Cas9 and inhibition of its degradation by small molecule NU7026. Asterisks indicate statistically significant differences: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

Analysis of StCas9 mismatch tolerance. a Schematic overview of generated St4 mutant gRNAs with nucleotide mismatches at the positions indicated on the bottom. In all cases, the nucleotide was changed to a complementary one to preserve CG composition. Effects of nucleotide mismatches on anti-HBV activity were analyzed by PCR measuring b, c HBV transcription, d HBV DNA and e cccDNA levels. Black squares indicate mutations at labeled sites. pgRNA/S-RNA levels were normalized to StCas9 mRNA, and HBV DNA/cccDNA levels were normalized to β-globin. ⁺p < 0.05, ^#p < 0.01, ^~p < 0.001, *p < 0.0001

Fig. 7

Deep sequencing of predicted off-target regions in the host genome for StCas9 and SpCas9. Frequency of indel mutations is indicated for a SpCas9 and b StCas9. Y-axis indicates off-target sites. Blue bars indicate mutation frequencies in mock-treated samples (Control), red bars indicate mutation frequencies post-transfection of HBV-targeting CRISPR/Cas9. ****p < 0.0001