Targeting Hepatitis B Virus With CRISPR/Cas9

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To determine whether IFN-α could enhance the antiviral activity exhibited by the CRISPR/Cas9 system, we treated NTCP/Cas9 cells expressing sg5 guide RNA 5 days after HBV infection for 72 hours with the cytokine (2,000 IU/ml). Under those conditions, IFN-α did not significantly reduce the fraction of HBcAg-positive cells when Cas9 was expressed (Figure 5). Similarly, we did not observe an increase in the fraction of mutations determined from the cccDNA analysis (Table 2), nor did we detect any additional types of mutations. However, IFN-α caused cell death at the high concentrations used in our experiments (2,000 and 1,000 IU/ml), in particular when cells were treated for 5 or 8 days with the cytokine (Supplementary Figure S3). Toxicity was even more apparent when the HBV-infected cells were treated with dox to induce Cas9.

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iscussion
We have developed a CRISPR/Cas9 platform for efficient inactivation of viral genes in NTCP expressing HepG2 cells permissive for HBV infection. We demonstrated that cccDNA can be targeted and inactivated as a consequence of Cas9-induced double-strand DNA breaks. While several previous reports demonstrated that transfected plasmid DNA corresponding to the HBV genome can be targeted by zinc fingers or transcription activator-like effector nucleases (TALENs), this is the first report to demonstrate that cccDNA formed from infectious virus can be cleaved and repaired like chromosomal DNA.30,31,32 Although not demonstrated experimentally in this report, this system can also be used for inactivation of cellular genes, which will enable future investigations on the role of host genes in the HBV life cycle.

While our results revealed that cccDNA derived from infectious virus is indeed accessible for Cas9 enzymatic cleavage, they also demonstrated that cleaved cccDNA is rapidly repaired, most likely by the NHEJ pathway normally used for repair of double-strand DNA breaks.

In this context, it is interesting to consider a previous report describing formation of a cccDNA species of the HBV-related duck hepatitis B virus from a double-stranded linear precursor.33 Such precursors are formed as a consequence of a so-called in situ priming reaction of plus strand DNA synthesis.34 While the nucleotide sequence of the joint DNA strands was not examined, DNA repair was dependent on Ku80 implying that NHEJ played a role as observed in our study with cccDNA derived from HBV rcDNA.

The nature of CRISPR/Cas9 mutations in cccDNA is similar to those observed with chromosomal DNA.35 The most frequent mutations (66%) were single-nucleotide deletions or insertions, followed by deletions that spanned over 100 nucleotides (19%) and smaller deletions of less than 30 nucleotides in length (12%). The reason for the vast difference in the size of the deletions spanning a few nucleotides to more than 2 kb is not known. One possibility might be that some cccDNA molecules are cleaved twice due to an off-target cleavage in addition to the target-specific event.36,37 However, considering that we observed large deletions with three different sgRNAs examined suggest that this is an unlikely explanation. However, it appears that sequences between positions 7 and 12 near DR1are preferred targets for the second cleavage site observed with sg5 and sg6 guide RNAs, respectively (Table 3 and Figure 1b). Perhaps, in rare cases, Cas9 cleavage might occur prior to cccDNA formation on rcDNA, leading to ligation of the 3′ end of minus strand DNA to the 5′ end of the cas9 cleaved minus strand DNA.

With sg5 RNA, we found a very good correlation between the sgRNA-induced inhibition of HBcAg expression and the ratio of wild-type and mutated clones obtained from PCR-amplified cccDNA (Figure 3 and Table 2). A described above, the most frequent mutations observed were single-nucleotide insertions and deletions that resulted in truncation of HBx and also in a change in the overlapping ENII/CP sequence motif (Figure 4). Therefore, it is not possible to determine the cause for the observed inhibition of HBcAg expression with certainty. We favor the conclusion that impairment of HBx expression is the primary cause for this inhibition, because previous studies have demonstrated that HBx activity is required for transcription of preC/C RNA from episomal cccDNA templates.38,39,40 Moreover, we consider it unlikely that different single-nucleotide deletions or insertions would significantly abrogate the activity of the ENII/PC regulator sequence. However, proof for this conclusion would require demonstration that the mutants can be rescued with wild-type HBx in trans. Attempts to transfect HBV-infected NTCP/Cas9 cells with an HBx expression plasmid have failed so far, because the DNA transfection efficiency was too low to detect a possible rescue of HBcAg expression. Efforts to improve the conditions for transfection are in progress.

Because a goal of this study was to investigate the stability of cccDNA following endonucleolytic cleavage, we also investigated whether activation of an antiviral innate immune response could augment the antiviral effect of the CRISPR/Cas9 system in conjunction with NHEJ. In an initial effort, we tested whether treatment of cells with IFN-α could further reduce the fraction of HBcAg-positive cells following HBV infection and sgRNA expression. These experiments were also motivated by a recent report claiming that IFN-α and tumor necrosis factor α could activate APOBEC 3A and B leading to enzymatic digestion of cccDNA.21 In contrast to the previous report, where HepaRG and primary hepatocyte cultures cells were treated for 10 days or more with the cytokines, in our experiments, IFN-α was added to HepG2 cells for only 3 days before cells were analyzed for HBcAg expression or harvested for cccDNA analysis. Moreover, we did not use 3D-PCR, a nested PCR reaction designed to selectively amplify DNA with a high A/T content, which can be the result of APOBEC-mediated deamination of C residues in rc or cccDNA.21,41 Nevertheless, under our selected conditions, we did not obtain any evidence for an enhanced antiviral activity against HBV in the presence or absence of sgRNAs. Moreover, we did not detect differences in the nature of the mutations in PCR clones derived from cccDNA. We observed some toxicity associated with IFN-α treatment and induction of Cas9 in HBV-infected NTCP/Cas9 cells (Supplementary Figure S3). It might be due to the combination of HBV infection with the CRISPR/Cas9 platform. Additional experiments will be required to investigate the activities of IFNs and other cytokines in this system. Finally, it might be possible to identify sgRNAs targeting additional regions on the HBV genome that might inactivate cccDNA with a significantly higher efficiency than we observed so far with our experiments.

While our results suggest that mutated cccDNA, even with substantial deletions, is relatively stable in HepG2 cells during the 7–10-day observation period used in our studies, they do not preclude the possibility that mutated cccDNA is lost more rapidly than wild-type DNA over longer time intervals spanning weeks and even months. Moreover, additional information about the exact timing of cccDNA formation and subsequent cleavage by Cas9 might help to obtain better estimates for the half-lives of mutated cccDNA. Whatever, the stability of mutated cccDNA might be, the fact that it is functionally inactivated as a consequence of the NHEJ emphasizes the potential of this repair pathway for an antiviral strategy. Although this study provided encouraging results toward a major goal in antiviral therapy against CHB, which is the therapeutic elimination of cccDNA from the liver, it leads to the more challenging issue concerning utility in the clinical setting. We favor a strategy based on small molecules that can either recruit a cellular endonuclease to cccDNA leading to repair by NHEJ. Such a strategy would require identification of a chemical structure that can recognize a specific motif on the cccDNA minichromosome. Perhaps subtle changes in histone spacing or a combination of host and virus proteins that bind to cccDNA can be exploited to achieve this goal

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Materials and methods
Cells. HepG2/NTCP cells were produced by infection of HepG2 cells with lentivirus NT-GFP; GFP expressing cells were enriched with the help of a FACS. NTCP/Cas9 cells were derived from HepG2/NTCP cells following infection with lentivirus vector pCW-Cas927 and selection with puromycin (1 µg/ml).

Antibodies. Antibodies against the myc-tags and FLAG tags were purchased from Cell Signaling Technology (Danvers, MA) and Sigma-Aldrich (St.Louis, MO), respectively. HBcAg antibody C1-5 was obtained from Santa Cruz Biotechnology (Dallas, Tx).

Vectors. For the construction of lentivirus vector NT-GFP, the NTCP gene obtained from Origene (Rockville, MD) was transferred into lentivirus vector pLVX-IRES-ZsGreen (Clontech, Mountain View, Ca). For the construction of pLX-SG1, a NdeI–AgeI fragment in pLX-AAVS1 sgRNA27 was replaced with a NdeI–AgeI fragment carrying two BsmBI/Esp3L restriction sites for cloning of guide RNA sequences with DNA oligomers (Supplementary Table S1). The nucleotide sequence of the NdeI–AgeI fragment in pLX-SG1 is shown in Supplementary Figure S1. sgRNA selection was based on algorithms developed by the Zhang group (crispr.mit.edu).

Virus and infections. HBV was concentrated 100-fold from the culture medium of HepAD38 cells with 6% polyethylene glycol and resuspended in serum-free Dulbecco's modified Eagle medium (DMEM)/F12 medium. Infection of HepG2 cells occurred in the presence of 4% polyethylene glycol in complete serum-free medium (DMEM/F12, pyruvate, nonessential amino acids, and penicillin/streptomycin) containing 2% dimethyl sulfoxide (DMSO) with an estimated 50 genome equivalents per cell. Medium was replaced with complete medium containing 10% fetal calf serum and 2% DMSO 24 hours after infection. Cultures were incubated for 5–10 days.

Isolation of cccDNA and rcDNA. HBV cccDNA was isolated from infected cells by the Hirt procedure.42 Briefly, cells from a well of a 12-well plate were lysed in 0.45 ml of sodium dodecyl sulfate (SDS) lysis buffer (50 mmol/l Tris-HCl pH7.5, 10 mmol/l EDTA, 0.5% SDS) for 10 minutes at 37 °C. One hundred microliter of a solution of 2.5 mol/l KCl (0.5 mol/l KCl final) was added and incubated at room temperature for 30 minutes to precipitate proteins and chromosomal DNA. After centrifugation at 10,000×g for 10 minutes, the Hirt supernatant was extracted twice with phenol and once with butanol:isopropanol (7:3). DNA was precipitated with two volumes of ethanol at room temperature for 2 hours and resuspended in 30 µl TE (10mmol/l Tris-HCl, 1 mmol/l EDTA). To increase the efficiency of the PCR reaction, the isolated cccDNA was linearized with EcoRI.

HBV rcDNA was isolated from the cytoplasm of HBV-infected cells as described by Yang et al.43 Briefly, cells from a well of a 12-well plate were lysed in 0.5 ml core lysis buffer (50 mmol/l Tris-HCl, pH7.5, 1 mmol/l EDTA, 1% NP40) for 10 minutes on ice. After removal of cell debris and nuclei by centrifugation, the cleared supernatant was incubated in a buffer containing 0.5% SDS, 10 mmol/l EDTA, 100 mmol/l NaCl, and 400 µg/ml pronase for 1 hour at 37 °C. DNA was extracted with phenol and butanol:isopropanol and then precipitated with ethanol at −20 °C for 2 hours. After centrifugation, the pellet containing core DNA was resuspended in 30 µl TE.

Immunofluorescence. For immunostaining, cells were fixed in 96-well plates with 4% paraformaldehyde for 10 minutes and processed for immunofluorescence with HBcAg monoclonal antibody C1-5 (Santa Cruz Biotechnology, Dallas, TX). The fraction of HBcAg-positive cells was determined with an ImageXpress Micro automated microscope (Molecular Devices Sunnyvale, Ca). Images from 16 preset positions at ×10 magnification with two channels (DAPI, Cy5) were collected from each 96 wells. Images were analyzed with MetaXpress imaging and analysis software using the multi-wavelength cell scoring module.

Statistical analysis. Unpaired, one-tailed t-tests (Prism 5) were used for the statistical analyses of the results shown in Figures 3b and 5.

SUPPLEMENTARY MATERIAL
Figure S1. Nucleotide sequence of NdeI-to-AgeI fragment in pLX-SG1.
Figure S2. HepAD38 cells (Ladner et al. 1997) were infected with lentivirus vectors expressing the respective guide RNAs.
Figure S3. Toxicity of IFN-α.
Table S1. Nucleotide sequence of DNA oligomers.

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References
Liaw, YF and Chu, CM (2009). Hepatitis B virus infection. Lancet 373: 582–592. | Article | PubMed | ISI | CAS |
Lozano, R, Naghavi, M, Foreman, K, Lim, S, Shibuya, K, Aboyans, V et al. (2012). Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380: 2095–2128. | Article | PubMed | ISI |
Trépo, C, Chan, HL and Lok, A (2014). Hepatitis B virus infection. Lancet (epub ahead of print).
Seeger, C and Mason, WS (2000). Hepatitis B virus biology. Microbiol Mol Biol Rev 64: 51–68. | Article | PubMed | ISI | CAS |
Seeger, C, Zoulim, F and Mason, WS (2013). Hepadnaviruses. In: Knipe, DM, Howley, P (eds). Lippincott, Williams and Wilkins: Philadelphia, PA Fields Virology 6th edition pp.2185-2221.
Sohn, JA, Litwin, S and Seeger, C (2009). Mechanism for CCC DNA synthesis in hepadnaviruses. PLoS One 4: e8093. | Article | PubMed |
Newbold, JE, Xin, H, Tencza, M, Sherman, G, Dean, J, Bowden, S et al. (1995). The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes. J Virol 69: 3350–3357. | PubMed | ISI | CAS |
Summers, J and Mason, WS (1982). Replication of the genome of a hepatitis B–like virus by reverse transcription of an RNA intermediate. Cell 29: 403–415. | Article | PubMed | ISI | CAS |
Seeger, C, Ganem, D and Varmus, HE (1986). Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232: 477–484. | Article | PubMed | ISI | CAS |
Tuttleman, JS, Pourcel, C and Summers, J (1986). Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 47: 451–460. | Article | PubMed | ISI | CAS |
Lenhoff, RJ and Summers, J (1994). Construction of avian hepadnavirus variants with enhanced replication and cytopathicity in primary hepatocytes. J Virol 68: 5706–5713. | PubMed | ISI |
Moraleda, G, Saputelli, J, Aldrich, CE, Averett, D, Condreay, L and Mason, WS (1997). Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol 71: 9392–9399. | PubMed | ISI | CAS |
Zhu, Y, Yamamoto, T, Cullen, J, Saputelli, J, Aldrich, CE, Miller, DS et al. (2001). Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 75: 311–322. | Article | PubMed | ISI |
Guo, JT, Pryce, M, Wang, X, Barrasa, MI, Hu, J and Seeger, C (2003). Conditional replication of duck hepatitis B virus in hepatoma cells. J Virol 77: 1885–1893. | Article | PubMed | ISI |
Reaiche-Miller, GY, Thorpe, M, Low, HC, Qiao, Q, Scougall, CA, Mason, WS et al. (2013). Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver. Virology 446: 357–364. | Article | PubMed | ISI |
Guidotti, LG, Rochford, R, Chung, J, Shapiro, M, Purcell, R and Chisari, FV (1999). Viral clearance without destruction of infected cells during acute HBV infection. Science 284: 825–829. | Article | PubMed | ISI | CAS |
Murray, JM, Wieland, SF, Purcell, RH and Chisari, FV (2005). Dynamics of hepatitis B virus clearance in chimpanzees. Proc Natl Acad Sci USA 102: 17780–17785. | Article | PubMed | CAS |
Kajino, K, Jilbert, AR, Saputelli, J, Aldrich, CE, Cullen, J and Mason, WS (1994). Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte. J Virol 68: 5792–5803. | PubMed | ISI | CAS |
Guo, JT, Zhou, H, Liu, C, Aldrich, C, Saputelli, J, Whitaker, T et al. (2000). Apoptosis and regeneration of hepatocytes during recovery from transient hepadnavirus infections. J Virol 74: 1495–1505. | Article | PubMed | ISI | CAS |
Summers, J, Jilbert, AR, Yang, W, Aldrich, CE, Saputelli, J, Litwin, S et al. (2003). Hepatocyte turnover during resolution of a transient hepadnaviral infection. Proc Natl Acad Sci USA 100: 11652–11659. | Article | PubMed |
Lucifora, J, Xia, Y, Reisinger, F, Zhang, K, Stadler, D, Cheng, X et al. (2014). Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343: 1221–1228. | Article | PubMed | ISI |
Chisari, FV, Mason, WS and Seeger, C (2014). Virology. Comment on “Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA”. Science 344: 1237. | Article | PubMed |
Yan, H, Zhong, G, Xu, G, He, W, Jing, Z, Gao, Z et al. (2012). Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1: e00049. | Article | PubMed | CAS |
Seeger, C and Mason, WS (2013). Sodium-dependent taurocholic cotransporting polypeptide: a candidate receptor for human hepatitis B virus. Gut 62: 1093–1095. | Article | PubMed | ISI |
Ran, FA, Hsu, PD, Wright, J, Agarwala, V, Scott, DA and Zhang, F (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8: 2281–2308. | Article | PubMed | ISI | CAS |
Hsu, PD, Lander, ES and Zhang, F (2014). Development and applications of CRISPR-Cas9 for genome engineering. Cell 157: 1262–1278. | Article | PubMed | ISI | CAS |
Wang, T, Wei, JJ, Sabatini, DM and Lander, ES (2014). Genetic screens in human cells using the CRISPR-Cas9 system. Science 343: 80–84. | Article | PubMed | ISI | CAS |
Ladner, SK, Otto, MJ, Barker, CS, Zaifert, K, Wang, GH, Guo, JT et al. (1997). Inducible expression of human hepatitis B virus (HBV) in stably transfected hepatoblastoma cells: a novel system for screening potential inhibitors of HBV replication. Antimicrob Agents Chemother 41: 1715–1720. | PubMed | ISI |
Guschin, DY, Waite, AJ, Katibah, GE, Miller, JC, Holmes, MC and Rebar, EJ (2010). A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649: 247–256. | PubMed | ISI | CAS |
Bloom, K, Ely, A, Mussolino, C, Cathomen, T and Arbuthnot, P (2013). Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol Ther 21: 1889–1897. | Article | PubMed | ISI | CAS |
Weber, ND, Stone, D, Sedlak, RH, De Silva Feelixge, HS, Roychoudhury, P, Schiffer, JT et al. (2014). AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS One 9: e97579. | Article | PubMed |
Cradick, TJ, Keck, K, Bradshaw, S, Jamieson, AC and McCaffrey, AP (2010). Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol Ther 18: 947–954. | Article | PubMed | ISI | CAS |
Guo, H, Xu, C, Zhou, T, Block, TM and Guo, JT (2012). Characterization of the host factors required for hepadnavirus covalently closed circular (ccc) DNA formation. PLoS One 7: e43270. | Article | PubMed |
Staprans, S, Loeb, DD and Ganem, D (1991). Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol 65: 1255–1262. | PubMed | ISI |
Cho, SW, Kim, S, Kim, JM and Kim, JS (2013). Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31: 230–232. | Article | PubMed | ISI | CAS |
Fu, Y, Foden, JA, Khayter, C, Maeder, ML, Reyon, D, Joung, JK et al. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31: 822–826. | Article | PubMed | ISI | CAS |
Hsu, PD, Scott, DA, Weinstein, JA, Ran, FA, Konermann, S, Agarwala, V et al. (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31: 827–832. | Article | PubMed | ISI | CAS |
Belloni, L, Pollicino, T, De Nicola, F, Guerrieri, F, Raffa, G, Fanciulli, M et al. (2009). Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci USA 106: 19975–19979. | Article | PubMed |
Lucifora, J, Arzberger, S, Durantel, D, Belloni, L, Strubin, M, Levrero, M et al. (2011). Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol 55: 996–1003. | Article | PubMed | ISI |
van Breugel, PC, Robert, EI, Mueller, H, Decorsière, A, Zoulim, F, Hantz, O et al. (2012). Hepatitis B virus X protein stimulates gene expression selectively from extrachromosomal DNA templates. Hepatology 56: 2116–2124. | Article | PubMed | ISI |
Suspène, R, Guétard, D, Henry, M, Sommer, P, Wain-Hobson, S and Vartanian, JP (2005). Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc Natl Acad Sci USA 102: 8321–8326. | Article | PubMed | CAS |
Hirt, B (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26: 365–369. | Article | PubMed | ISI | CAS |
Yang, W, Mason, WS and Summers, J (1996). Covalently closed circular viral DNA formed from two types of linear DNA in woodchuck hepatitis virus-infected liver. J Virol 70: 4567–4575. | PubMed | ISI |
Levin, BC, Cheng, H and Reeder, DJ (1999). A human mitochondrial DNA standard reference material for quality control in forensic identification, medical diagnosis, and mutation detection. Genomics 55: 135–146. | Article | PubMed | ISI |
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Acknowledgements
We thank Sid Balachandran and Richard Katz for helpful comments to the manuscript and Linghuan Tang for excellent technical assistance. We acknowledge services provided by the following Fox Chase Cancer Center facilities: High throughput, Imaging and DNA sequencing. C.S. acknowledges support from the National Institutes of Health grants AI103514 and AI101558 and the Commonwealth of Pennsylvania.

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