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Cited by 386Hepatitis B Virus StudiesCRISPR and Genetic EngineeringHepatitis C virus research

The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo

Open access
Lin, Su-Ru, Yang, Hung-Chih, Kuo, Yi-Ting, Liu, Chun-Jen, Yang, Ta-Yu, Sung, Ku-Chun, Lin, You-Yu, Wang, Hurng-Yi +6 more·2014·Molecular Therapy - Nucleic Acids
DOI 10.1038/mtna.2014.38PMC PMC4221598
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Abstract

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

AI snapshot

Written from the paper’s full text.

TL;DR

The CRISPR/Cas9 system with HBV-specific gRNAs reduced HBV protein expression in Huh7 cells and cleaved intrahepatic HBV templates in a hydrodynamic mouse model, lowering serum HBsAg. A conserved gRNA (XCp) worked across HBV genotypes A/B/C, and multiplex gRNAs increased cleavage efficiency.

Key findings

  • Eight gRNAs were designed against HBV genotype A; in Huh7 cells, gRNAs P1, S1, PS2, and PS3 suppressed intracellular HBV core/surface protein expression by 70%, 40%, 30%, and 20%, respectively. Dual gRNA/Cas9 vectors with P1, S1, XCp, and PS2 suppressed intracellular surface proteins by 96%, 90%, 82%, and 77%; supernatant HBsAg was suppressed by P1, XCp, and PS2 by 64%, 36%, and 16%, while S1 failed to reduce extracellular HBsAg.
  • gRNA XCp, targeting a conserved HBV sequence, was equally effective against genotypes A, B, and C; gRNA P1 did not significantly suppress genotype B, likely because genotypes B and C have a single-nucleotide polymorphism in the PAM despite matching the 20-bp target.
  • Multiplex gRNAs P1 + XCp suppressed intracellular HBsAg more than either alone, though marginally significant (P = 0.078 vs P1; P = 0.085 vs XCp); T7E1 showed Indel rates of 13.6% for P1, 9.3% for XCp, and 25.6% for the combination, with a ~580-bp cleaved DNA segment observed.
  • In hydrodynamics-injected C57BL/6 mice, serum HBsAg was significantly lower on day 2 with P1 and XCp, and on day 6 only with P1; intrahepatic HBV-expressing vector levels were reduced by Southern blot, with T7E1 targeted disruption in 5% (P1) and 4% (XCp) of intrahepatic HBV genomes, and clonal sequencing found 5/18 clones (27.8%) with Indel for P1.

Methods

  • In vitro: Huh7 cells were cotransfected with the HBV-expression vector pAAV/HBV1.2 (genotype A) and individual or dual gRNA/Cas9 expression vectors; HBV proteins were measured by Western blot and HBsAg ELISA. A surface-deficient DHBV-expressing plasmid in 293T cells was used to assess rc-DNA and cccDNA by Southern blot.
  • In vivo: C57BL/6 mice (6–8 weeks old) received hydrodynamic tail-vein injection of 6 μg pAAV/HBV1.2 and 10 μg gRNA/Cas9 dual expression vector; serum HBsAg, intrahepatic HBV DNA (Hirt extraction, Southern blot), T7E1 assay, and clonal sequencing were performed.
  • gRNA design: eight HBV-specific gRNAs were selected with a 5′ G and downstream PAM (GN19-NGG); dual expression vectors contained both gRNA and Cas9 cassettes. Statistical comparisons used an unpaired, one-tailed Student’s t-test.

Limitations

  • The paper states that the HBV hydrodynamics-mouse model is not an HBV infection model and no cccDNA is detectable; it remains unclear whether episomal cccDNA can be disrupted efficiently, and animal models with bona fide cccDNA (human–mouse hepatocyte chimeric or WHV-infected woodchuck) are still required.
  • The paper states that efficiency can be improved, residual serum HBsAg persisted after treatment, off-target effects of the gRNAs cannot be excluded, and the effect of cleaving integrated HBV genomes on host genome stability should be carefully evaluated.
  • Evident from the text: in the HBV cell-culture system, the HBV-expressing plasmid generated very few cccDNA, if any, so the DHBV system was used as a surrogate for cccDNA studies.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/mtna.2014.38

Cites

40 of 40 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 29 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Levels of intrahepatic HBV-expressing vectors were significantly reduced in mice receiving HBV-specific gRNAs P1 and XCp.

    “In addition, using Southern blot analysis, we found that the levels of intrahepatic HBV-expressing vectors, an indicator for persistent HBV genomes, were significantly reduced in those mice receiving HBV-specific gRNAs P1 and XCp (Figure 5b).”
    Quote in the paperSubstantial claim
  • Findingown data

    T7E1 assay showed target-specific mutagenesis in mice receiving gRNA-P1 or -XCp/Cas9 dual expression vector, but not in control mice.

    “To confirm that this CRISPR/Cas9 system specifically cleaves the target sequences, we performed the T7E1 assay, which showed target-specific mutagenesis in mice receiving the gRNA-P1 or -XCp/Cas9 dual expression vector, but not in control mice (Figure 5c).”
    Quote in the paperSubstantial claim
  • Findingown data

    The HBV-specific gRNAs/Cas9 reduced the intrahepatic HBV templates.

    “Finally, we demonstrated by Southern blot analysis that the HBV-specific gRNAs/Cas9 reduced the intrahepatic HBV templates.”
    Quote in the paperSubstantial claim
  • Findingown data

    Among eight screened gRNAs, two effective ones were identified.

    “Among eight screened gRNAs, two effective ones were identified.”
    Quote in the paperNot a substantial claim
  • Findingown data

    A shorter band (~580 bp) in the PCR product from cells treated with combinatorial gRNAs P1 and XCp proved by sequencing to be the cleaved DNA segment mediated by gRNAs P1 and XCp.

    “Interestingly, we also observed a shorter band (~580 bp) in the PCR product directly amplified from cells treated with combinatorial gRNAs P1 and XCp (Supplementary Figure S2).”
    • Also stated in: § Multiplex gRNAs exhibited stronger effects on suppressing the expression of HBV proteins · plain
      “By sequencing, the band proved to be the cleaved DNA segment mediated by gRNAs P1 and XCp (data not shown).”
    Quote in the paperSubstantial claim
  • May combine two claims. 27
  • Classified as probably not a substantial claim. 12

  • May combine two claims.

    Findingown data

    Five of 18 clones (27.8%) from mice receiving gRNA P1 had Indel, suggesting NHEJ repair occurred at the cleaved target sequence of gRNA P1.

    “Five clones (27.8%) with Indel were noted, suggesting that nonhomologous end joining (NHEJ) repair has occurred at the cleaved target sequence of gRNA P1 (Supplementary Figure S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combinatorial gRNAs P1 and XCp were more effective in suppressing intracellular HBsAg production than either gRNA alone, although the effect is only marginally significant (P = 0.078 for combination versus P1 and P = 0.085 for combination versus XCp).

    “The results demonstrated that the combinatorial gRNAs P1 and XCp were more effective in suppressing intracellular HBsAg production than either gRNA alone, although the effect is only marginally significant (P = 0.078 for combination versus P1 and P = 0.085 for combination versus XCp) (Figure 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the hydrodynamics system, serum HBsAg was reduced but still persisted following CRISPR/Cas9 treatment.

    “In our hydrodynamics system, serum HBsAg was reduced, but still persisted following CRISPR/Cas9 treatment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Serum HBsAg levels indicate the persistence of HBV.

  • May combine two claims.

    Findingown data

    gRNA S1 significantly suppressed intracellular HBsAg and HBcAg but failed to reduce extracellular HBsAg levels.

    “Surprisingly, although the gRNA S1 significantly suppressed the expression of intracellular HBsAg and HBcAg, it failed to reduce the extracellular HBsAg levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Using a hydrodynamics-HBV persistence mouse model, the CRISPR/Cas9 system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels.

    “Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Serum HBsAg levels indicate the persistence of HBV.

  • May combine two claims.

    Findingown data

    The most effective gRNA P1 against genotype A did not significantly suppress viral protein expression of genotype B, although it significantly suppressed that of genotype C.

    “In contrast, the most effective gRNA P1 against genotype A of HBV did not significantly suppress the viral protein expression of genotype B, although it significantly suppressed that of genotype C (Figure 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percentage of Indel mediated by combinatorial gRNAs P1 and XCp was 25.6%, higher than that mediated by P1 or XCp alone.

    “Besides, the percentage of Indel mediated by combinatorial gRNAs P1 and XCp was 25.6%, which was higher than that mediated by P1 or XCp alone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HBV genomes of genotypes B and C had a single-nucleotide polymorphism in the PAM for gRNA P1, although they had the perfectly matched 20-bp target sequence.

    “Analysis of the target sequences of gRNA P1 in the HBV genomes of these three genotypes revealed that the HBV genomes of genotypes B and C had a single-nucleotide polymorphism in the PAM, although they had the perfectly matched 20-bp target sequence (Figure 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The use of combinatorial gRNAs P1 and XCp exhibited slightly higher efficiency in reducing HBV protein production than use of either individual gRNA alone and generated a larger fragment of cleaved DNA.

    “As demonstrated here, the use of combinatorial gRNAs P1 and XCp exhibited slightly higher efficiency in reducing the HBV protein production than use of either individual gRNA alone and generated a larger fragment of cleaved DNA, exemplifying the advantage of utilizing multiplex gRNAs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DHBV-specific gRNAs slightly reduced levels of DHBV-expressing plasmid but efficiently suppressed levels of rc-DNA and cccDNA.

    “Compared with the mock gRNA-transfection control, the DHBV-specific gRNAs slightly reduced the levels of DHBV-expressing plasmid, as demonstrated by Southern blot, but efficiently suppressed the levels of the rc-DNA and cccDNA (Supplementary Figure S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    gRNAs P1, XCp, and PS2 suppressed HBsAg levels in culture supernatant by 64, 36, and 16%, respectively.

    “The gRNAs P1, XCp, and PS2 suppressed the HBsAg levels by 64, 36, and 16%, respectively (Figure 2c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The occurrence of mutated HBV expression templates edited by gRNAs P1 and XCp alone was 13.6 and 9.3%, respectively.

    “The results indicated that the occurrence of mutated HBV expression templates edited by gRNAs P1 and XCp alone was 13.6 and 9.3%, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    gRNAs P1, S1, PS2, and PS3 exhibited higher efficacy in suppressing expression of HBV proteins by 70, 40, 30, and 20%, respectively.

    “we found that the gRNAs P1, S1, PS2, and PS3 exhibited higher efficacy in suppressing the expression of HBV proteins by 70, 40, 30, and 20%, respectively (Figure 2a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The suppressive effect of gRNA P1 on viral protein expression of genotype C is much less than that of gRNA XCp (P < 0.05), whereas both gRNAs exhibited a similar inhibitory effect on genotype A.

    “Nevertheless, the suppressive effect of gRNA P1 on viral protein expression of genotype C is much less than that of gRNA XCp (P < 0.05), whereas both gRNAs exhibited a similar inhibitory effect on genotype A.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Interpretationcitation

    Theoretically, combined with effective suppression of HBV replication by NAs, specific disruption of the HBV genome may result in eradication of HBV cccDNA, hence curing chronic HBV infection.

    “Theoretically, combined with effective suppression of HBV replication by NAs, specific disruption of the HBV genome may result in eradication of HBV cccDNA, hence curing chronic HBV infection.7”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Schiffer, 2013, Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach, PLoS Comput Biol. source

      Not checked in the free run.

    • Rests on an assumption: Curing chronic hepatitis B requires elimination of persistent HBV cccDNA or infected hepatocytes.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ZFNs and TALENs can cleave host genomes in a site-specific fashion and have been under investigation for specific degradation of HBV genomes in vitro and in vivo.

    “Two genome editing technologies, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), can cleave the host genomes in a site-specific fashion and have been under investigation for specific degradation of the HBV genomes in vitro and in vivo.8,9,10”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] Cradick, 2010, Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs, Mol Ther source

      Not checked in the free run.

    • cites [9] Bloom, 2013, Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

    • cites [10] Chen, 2014, An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    HBV cccDNA exhibits staggering stability and declines slowly under antiviral therapy, representing a major barrier to viral eradication.

    “Moreover, HBV cccDNA exhibits staggering stability and declines slowly under antiviral therapy, thus representing a major barrier to viral eradication.3,4,5,6”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [3] Tsiang, 1999, Biphasic clearance kinetics of hepatitis B virus from patients during adefovir dipivoxil therapy, Hepatology source

      Not checked in the free run.

    • cites [4] Werle–Lapostolle, 2004, Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy, Gastroenterology source

      Not checked in the free run.

    • cites [5] Caruntu, 2005, CccDNA persistence during natural evolution of chronic VHB infection, Rom J Gastroenterol

      Not checked in the free run.

    • cites [6] Sung, 2005, Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy, Gastroenterology source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The CRISPR/Cas system has been successfully applied to human cells and several model organisms for genome editing.

    “Recently, the CRISPR/Cas system has been successfully applied to human cells19,20,21,22 and several model organisms for genome editing.23,24,25,26”
    Quote in the paperNot a substantial claim0 of 8 sources checked
    • cites [19] Mali, 2013, RNA-guided human genome engineering via Cas9, Science source

      Not checked in the free run.

    • cites [20] Cong, 2013, Multiplex genome engineering using CRISPR/Cas systems, Science source

      Not checked in the free run.

    • cites [21] Cho, 2013, Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease, Nat Biotechnol source

      Not checked in the free run.

    • cites [22] Jinek, 2013, RNA-programmed genome editing in human cells, Elife source

      Not checked in the free run.

    • cites [23] Friedland, 2013, Heritable genome editing in C. elegans via a CRISPR-Cas9 system, Nat Methods source

      Not checked in the free run.

    • cites [24] Hwang, 2013, Efficient genome editing in zebrafish using a CRISPR-Cas system, Nat Biotechnol source

      Not checked in the free run.

    • cites [25] Katic, 2013, Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans, Genetics source

      Not checked in the free run.

    • cites [26] Wang, 2013, One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering, Cell source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In CHB patients, up to 50 copies of cccDNA exist in each infected hepatocyte, and some of them are epigenetically modified.

    “In CHB patients, up to 50 copies of cccDNA exist in each infected hepatocyte, and some of them are epigenetically modified.34”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Levrero, 2009, Control of cccDNA function in hepatitis B virus infection, J Hepatol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    As a proof of concept, the CRISPR/Cas9 system has utility in disrupting intrahepatic HBV templates without apparent toxicity.

    “In conclusion, as a proof of concept, we have shown the utility of the CRISPR/Cas9 system in disrupting intrahepatic HBV templates without apparent toxicity.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The CRISPR/Cas9 system was also utilized to disrupt the latent integrated HIV provirus.

    “Besides, the CRISPR/Cas9 system was also utilized to disrupt the latent integrated HIV provirus.36”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Ebina, 2013, Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus, Sci Rep source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Any residual HBV genome can potentially lead to rebound viremia and repopulation of HBV in the liver after cessation of antiviral therapy.

    “Theoretically, any residual HBV genome can potentially lead to rebound viremia and repopulation of HBV in the liver after cessation of the antiviral therapy.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The CRISPR/Cas9 system has been used for multiplex genome cleavage.

    “The CRISPR/Cas9 system has been used for multiplex genome cleavage.20”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [20] Cong, 2013, Multiplex genome engineering using CRISPR/Cas systems, Science source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The importance of PAM for genome cleavage by the CRISPR/Cas9 system is indicated, and the effects of gRNAs P1 and XCp are mediated by the CRISPR/Cas9 system.

    “This result indicates the importance of PAM for genome cleavage by the CRISPR/Cas9 system, and also provides further evidence that the effects of gRNAs P1 and XCp are mediated by the CRISPR/Cas9 system.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    The off-target effects of the gRNAs cannot be excluded.

    “the off-target effects of our gRNAs cannot be excluded”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The HBV hydrodynamics-mouse model is a well-established model of HBV persistence.

    “The HBV hydrodynamics-mouse model is a well-established model of HBV persistence.28,29”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [28] Huang, 2006, An immunocompetent mouse model for the tolerance of human chronic hepatitis B virus infection, Proc Natl Acad Sci USA. source

      Not checked in the free run.

    • cites [29] Li, 2013, Blocking the natural killer cell inhibitory receptor NKG2A increases activity of human natural killer cells and clears hepatitis B virus infection in mice, Gastroenterology source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The CRISPR/Cas9 system was as effective as TALENs in mediating gene disruption in vitro, although its mutagenesis rate is lower (~5% by T7E1 and ~27% by clonal sequencing) in vivo.

    “Our study demonstrated that the CRISPR/Cas9 system was as effective as TALENs in mediating gene disruption in vitro, although its mutagenesis rate is lower (~5% by T7E1 and ~27% by clonal sequencing) in vivo.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] Bloom, 2013, Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

    • cites [10] Chen, 2014, An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    A previous study showed that a 13-bp seed region at the 3′-end of the protospacer sequence is critical for site-specific cleavage, indicating that Cas9 can tolerate up to seven mismatches at the 5′-end of the 20-base protospacer sequence.

    “A previous study showed that a 13-bp “seed” region located at the 3′-end of the protospacer sequence is critical for its site-specific cleavage, indicating that Cas9 can tolerate up to seven mismatches at the 5′-end of the 20-base protospacer sequence.18”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [18] Jinek, 2012, A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Science source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Simultaneous cleavage of multiple sites can be achieved by using multiplex gRNAs that could increase the efficiency for disrupting HBV genomes and reduce the emergence of viral mutations escaping from cleavage.

    “Additionally, simultaneous cleavage of multiple sites can be achieved by using multiplex gRNAs that could increase the efficiency for disrupting HBV genomes and reduce the emergence of viral mutations escaping from cleavage.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductnone given

    The study used the HBV hydrodynamics-mouse model, which is not a model of HBV infection and has no detectable cccDNA.

    “One limitation of this study is the use of the HBV hydrodynamics-mouse model, which is not a model of HBV infection and no cccDNA is detectable.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The CRISPR/Cas9 system cleaved the HBV genome-expressing template and facilitated its clearance.

    “Taken together, our results indicated that the CRISPR/Cas9 system cleaved the HBV genome-expressing template and facilitated its clearance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous studies reported that HBV-specific TALENs have a mutagenesis rate ranging between 30 and 70%.

    “Previous studies reported that HBV-specific TALENs have a mutagenesis rate ranging between 30 and 70%.9,10”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [9] Bloom, 2013, Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

    • cites [10] Chen, 2014, An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases, Mol Ther source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The CRISPR/Cas9 system could disrupt HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

    “These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A very recent study reported that the CRISPR/Cas9 system could cleave the large-genome DNA viruses adenovirus and herpes simplex virus with high efficiency.

    “Interestingly, a very recent study reported that the CRSIPR/Cas9 system could cleave the large-genome DNA viruses, adenovirus and herpes simplex virus, with high efficiency.35”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Bi, 2014, High-efficiency targeted editing of large viral genomes by RNA-guided nucleases, PLoS Pathog source

      Not checked in the free run.

34 of 34

  • The paper relies on this without support.

    Curing chronic hepatitis B requires elimination of persistent HBV cccDNA or infected hepatocytes.

    “Curing CHB requires elimination of persistent HBV cccDNA or infected hepatocytes, which is so far still an unattainable goal.”

  • The paper relies on this without support.

    The CRISPR/Cas system can be easily reprogrammed to cleave virtually any DNA sequence by simply redesigning the crRNAs or gRNAs.

    “Compared with ZFNs and TALENs, the CRISPR/Cas system can be easily reprogrammed to cleave virtually any DNA sequence by simply redesigning the crRNAs or gRNAs.”

  • The paper relies on this without support.

    Serum HBsAg levels indicate the persistence of HBV.

    “The concentration of HBs antigen was determined by AXSYM systems (Abbott Park, IL) and indicated the persistence of HBV.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • CRISPR/Cas9 system holds tremendous promise for gene therapy and can correct causal mutations in the original genome and rescue disease phenotypes of monogenic disorders permanently.

    “Furthermore, CRISPR/Cas9 system holds tremendous promise for gene therapy [46–51]. It can correct causal mutations in the original genome and rescue the disease phenotypes of monogenic disorders permanently, which currently represents the most translatable field in CRISPR/Cas9-mediated disease therapy.”

    From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [48]

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Authors
Lin, S.-R., Yang, H.-C., Kuo, Y.-T., Liu, C.-J., Yang, T.-Y., Sung, K.-C., Lin, Y.-Y., Wang, H.-Y., Wang, C.-C., Shen, Y.-C., Wu, F.-Y., Kao, J.-H., Chen, D.-S., & Chen, P.-J.
Affiliations
National Taiwan University (TW)
Corresponding author
Yang, Hung-Chih
ORCID
Venue
Molecular Therapy - Nucleic Acids
Publisher
Elsevier BV
Published
2014-01
Volume
3
Pages
e186
ISSN
2162-2531 (ISSN-L)
DOI
10.1038/mtna.2014.38
PMC
PMC4221598
OpenAlex
W2056607224
Semantic Scholar
cf6222971e043692b30a692330fc234210f794bb
Cited by
386
References
40
Citations by year
2026: 7 · 2025: 12 · 2024: 14 · 2023: 24 · 2022: 29 · 2021: 28 · 2020: 36 · 2019: 31 · 2018: 44 · 2017: 58 · 2016: 56 · 2015: 42 · 2014: 5
FWCI
40.55
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Taiwan University Hospital (103-P01)
Topics
  • Health Sciences › Medicine › Epidemiology › Hepatitis B Virus Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Hepatology › Hepatitis C virus research
Keywords
CRISPR/Cas9, hepatitis B virus, covalently closed circular DNA, chronic hepatitis B, guide RNA design, HBV genotype A, hepatitis B surface antigen, Huh7 cells, antiviral gene therapy

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