Materials and Methods

CRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants

Cell lines, viral inoculum, and infection conditions.

HBV particles were concentrated from the supernatant of HepAD38 cells (HBV genotype D; kind gift from C. Seeger Fox Chase Cancer Center, USA) (36) by filtering and PEG precipitation (37). HepG2-NTCP cells (kind gift from S. Urban Heidelberg University, Germany) (38) were seeded at 105 cells/cm2 in growth medium (Dulbecco’s modified Eagle medium DMEM; high glucose, 1% penicillin-streptomycin, 1% sodium pyruvate, 1% glutamine Life Technologies, 5% fetal calf serum HyClone fetal clone II). From the next day onward, cells were cultured in medium complemented with 2.5% dimethyl sulfoxide (DMSO) (Sigma) as a standard method to increase HBV infection without affecting cell viability (39). After 72 h, cells were infected at a multiplicity of infection of 1,000 (4% PEG, 16 h). For protocol 1, infected cells were replated at 3 dpi at 5 × 104 cells/cm2. At 4 dpi, cells were transfected with RNPs, and medium was replaced 24 h posttransfection (hpt). Supernatants and cells lysates were collected at 7 dpi (4 dpt) or 14 dpi (10 dpt). When indicated, cells were treated with 3TC (10 μM) from 4 dpi until cell collection. In protocol 1, for the “rebound control with or without (+/−) 3TC” condition, 3TC was present for 3 days and then removed for a week. For protocol 2, 3TC was added at 4 dpi and cells were replated at 6 dpi at 8 × 104 cells/cm2 in the continuous presence or absence of 3TC.

gRNA design, RNP assembly, and transfection.

The CRISPR RNA (crRNA) sequences for Sp1 to Sp8 were adapted from published studies (40,–42), while Sp9 to Sp12 gRNA sequences were designed using the online Benchling platform minimizing homology to the human genome. crRNAs, Neg1 and -2, trans-activating crRNA (tracrRNA)-ATTO550, and Streptococcus pyogenes (S.py) Cas9 protein (WT and dead) were purchased from IDT-DNA. gRNAs were in vitro assembled by mixing equimolar concentrations of crRNA and tracrRNA (with or without ATTO550, as indicated) at a final concentration of 30 nM, with heating and cooling down at room temperature to allow formation of heteroduplexes. JetCRISPR (PolyPlus transfection) was used for forward transfection in infected HepG2-NTCP cells.

Real-time PCRs.

RNA and DNA were extracted using the Macherey-Nagel NucleoSpin RNA kit and the Epicentre MasterPure kit, respectively. Real-time PCRs were performed as previously described (43). The 3.5-kb RNA was normalized to the housekeeping gene beta-glucuronidase (GUSB) (Hs99999908m1; Thermo Fisher Scientific). Total HBV DNA was measured using TaqMan assay Pa03453406_s1 (Life Technologies). For cccDNA quantification, incomplete double-stranded circular DNA was degraded by ExoI+III treatment, and primers targeting a specific region in cccDNA were used (44) (forward, 5′_CCGTGTGCACTTCGCTTCA_3′; reverse, 5′_GCACAGCTTGGAGGCTTGA3′; probe, 5′6FAMCATGGAGACCACCGTGAACGCCCBBQ). Serial dilutions of an HBV plasmid served as an external quantification standard. Total DNA or cccDNA was normalized to human beta globin (HBB) (Hs00758889_s1; Thermo Fisher Scientific).

ELISAs for viral antigens.

Enzyme-linked immunosorbent assays (ELISAs) for HBeAg and HBsAg detection in cell supernatants were performed according to the manufacturer’s protocol using the chemiluminescence immunoassay (CLIA) kit from Autobio Diagnostic. All measurements were performed after 3 days of accumulation in medium.

Southern blotting.

Cells (2 × 107) were processed using a modified Hirt extraction followed by ExoI+III digestion (45). Samples were then quantified by Qubit, and ND2 qPCR was used to normalize the seeding amount to mitochondrial DNA. Samples were run in a 1.2% agarose gel for 16 h at 15 V. Samples in the agarose gel were depurinated using HCl followed by NaOH treatment and neutralization before transfer to a nylon membrane using a Whatman Turbo blotter. Membranes were blocked and incubated with branched-DNA probes to detect the negative-strand HBV DNA with modifications of the protocol described in reference 46 or with digoxigenin (DIG) DNA probes as described in reference 47.

RNA sequencing.

RNA was extracted using TRIzol followed by Turbo I DNase treatment. An RNA integrity number of >9.6 was confirmed with a Bioanalyzer prior to submission to Genewiz. cDNA libraries were constructed using a NEBNext Ultra RNA library preparation kit with rRNA depletion. Paired-end sequencing was conducted using Illumina MiSeq with a read length of 150 bp (average of ∼100 × 106 reads/sample). Sequencing reads were aligned to the human and HBV reference genome (GenBank no. U95551.1) using STAR (48). To calculate the frequency, two variant callers (Pindel and HaplotypeCaller) were used to determine the number of reads supporting a given mutation. The ratio of mutations to the total number of reads (mutated or not) was then manually calculated using the data provided by the variant callers. For the CRISPR variants, custom reference genomes were generated based on the expected cleavage sites of S.py Cas9 (−3 from the PAM) followed by recircularization of each double-cleaved fragment. Coverage calculation per position was performed using GenomeCov from the toolkit bedtools. The original coverage per position in the genome ranged from 2,000 to 15,432.

Amplicon sequencing.

Nuclear extraction of 6 × 106 cells was performed in cell lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride PMSF, 1× protease inhibitor cocktail PIC; Thermo Fisher). After “tight” treatment with Dounce and centrifugation (10 min, 4,500 rpm), nuclei were resuspended in 1× Tris-EDTA (TE) buffer. Episomal DNA was enriched using a modified Hirt extraction (45), followed by phenol-chloroform DNA purification, ExoI+III digestion, and Zymo column cleanup. Primers pairs for nested PCR using HiFi Q5 polymerase were designed to flank Sp5 and Sp7 target sites for on-target amplicon sequencing. To calculate the frequency, two variant callers (Pindel and HaplotypeCaller) were used to determine the number of reads supporting a given mutation. The ratio of mutations to the total number of reads (mutated or not) was then manually calculated using the data provided by the variant callers. For the CRISPR variants, same primers were combined in different pairs around the novel predicted junctions. Amplicons of around 300 bp were then submitted for next-generation sequencing (NGS) to Genewiz facilities (Amplicon-EZ) (Fig. S8E).

Statistical analysis.

Statistical analysis was performed using Prism 7 software (GraphPad Software, San Diego, CA, USA). The Kruskal-Wallis test and Dunn’s multiple-comparison test were used to compare numerical data.

Data availability.

DNA amplicon-sequencing and RNA sequencing data are be available at the European Nucleotide Archive (ENA) under accession number PRJEB51625.

Article TitleCRISPR-Cas9 Targeting of Hepatitis B Virus Covalently Closed Circular DNA Generates Transcriptionally Active Episomal Variants

Abstract

Chronic hepatitis B virus (HBV) infection persists due to the lack of therapies that effectively target the HBV covalently closed circular DNA (cccDNA). We used HBV-specific guide RNAs (gRNAs) and CRISPR-Cas9 and determined the fate of cccDNA after gene editing. We set up a ribonucleoprotein (RNP) delivery system in HBV-infected HepG2-NTCP cells. HBV parameters after Cas9 editing were analyzed. Southern blot (SB) analysis and DNA/RNA sequencing (DNA/RNA-seq) were performed to determine the consequences of cccDNA editing and transcriptional activity of mutated cccDNA. Treatment of infected cells with HBV-specific gRNAs showed that CRISPR-Cas9 can efficiently affect HBV replication. The appearance of episomal HBV DNA variants after dual gRNA treatment was observed by PCR, SB analysis, and DNA/RNA-seq. These transcriptionally active variants are the products of simultaneous Cas9-induced double-strand breaks in two target sites, followed by repair and religation of both short and long fragments. Following suppression of HBV DNA replicative intermediates by nucleoside analogs, mutations and formation of smaller transcriptionally active HBV variants were still observed, suggesting that established cccDNA is accessible to CRISPR-Cas9 editing. Targeting HBV DNA with CRISPR-Cas9 leads to cleavage followed by appearance of episomal HBV DNA variants. Effects induced by Cas9 were sustainable after RNP degradation/loss of detection, suggesting permanent changes in the HBV genome instead of transient effects due to transcriptional interference.


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