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Cited by 148CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA regulation and disease

High-efficiency targeted editing of large viral genomes by RNA-guided nucleases

Open access
Bi, Yanwei, Sun, Le, Gao, Dandan, Ding, Chen, Li, Zhihua, Li, Yadong, Cun, Wei, Li, Qihan·2014·PLoS Pathogens
DOI 10.1371/journal.ppat.1004090PMC PMC4006927
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Abstract

A facile and efficient method for the precise editing of large viral genomes is required for the selection of attenuated vaccine strains and the construction of gene therapy vectors. The type II prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)) RNA-guided nuclease system can be introduced into host cells during viral replication. The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency. Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus. As a result, purified recombinant virus can be obtained with only a single round of selection. In this study, we used recombinant adenovirus and type I herpes simplex virus as examples to demonstrate that the CRISPR-Cas9 system is a valuable tool for editing the genomes of large DNA viruses.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper demonstrates that CRISPR-Cas9 RNA-guided nucleases can efficiently edit large DNA viral genomes, using recombinant adenovirus and HSV1 as models, by inducing NHEJ-mediated indels or HDR-mediated gene insertion/replacement. The approach also suppresses replication of the targeted wild-type virus, enriching recombinant progeny so that purified mutants can be isolated in a single round.

Key findings

  • In ADV-EGFP, three gRNAs induced site-specific mutations in the adenoviral genome; gRNA-175 was most efficient at 47.4%, followed by gRNA-174 at 37.3% and gRNA-173 at 32.7% by SURVEYOR assay. Mutations were heritable to P2 progeny, though mutant genomes were less likely than wild-type to form infectious particles.
  • Cas9:gRNA-175 inhibited ADV-EGFP replication: the logarithmic growth phase was delayed from 24–36 h to 36–48 h, and peak titer was 4–5-times lower than control. At 24 h post-infection, only 6.4±1.1% of viral genomes were intact, and mutant progeny reached 47%–52% of total genomes at 36 h.
  • HDR with donor DNA replaced ADV EGFP with DsRed at 2.6±0.57% efficiency, versus below 10−7.4 in controls; for HSV1, EGFP knock-in reached 8.41% efficiency, versus less than 1.45×10−8 in controls.
  • For HSV1 UL23/TK targeting, gRNA-206 produced >50% uncleaved fragments in RFLP; ACV-resistant progeny reached 3.16×10⁷ TCID50/ml, 50.1% of total virus, while total HSV1 titer decreased from 3.2×10⁸ to 6.31×10⁷ TCID50/ml with RGN. Deep sequencing found no off-target mutations in the tested viral homologous sites for gRNA-175 or gRNA-206.

Methods

  • Experimental design: CRISPR-Cas9 and Cas9n nickase systems were introduced into host cells before or during infection with recombinant adenovirus ADV-EGFP or HSV1 strain 8F; donor DNA was supplied for HDR-mediated gene replacement/insertion.
  • Cells and viruses: 293FT, AD293, and Vero cells were used; ADV-EGFP was cultured/titered on AD293 cells, and HSV1 strain 8F on Vero cells.
  • Main analyses: SURVEYOR nuclease assay, T-vector cloning/Sanger sequencing, RFLP, deep amplicon sequencing, qPCR for viral genome repair, plaque assays, endpoint dilution titration, fluorescence microscopy, and whole-genome sequencing of selected mutants.

Limitations

  • The paper states that high cleavage efficiency and low cellular repair efficiency may limit multiple-site editing of viral genomes within a single replication cycle, and that P1 mutant virus is not passed to P2 in equal proportions, possibly due to Cas9 steric hindrance or cleavage/repair timing affecting packaging.
  • The paper notes that because viral genomes are compact and genes overlap, a single cleavage may sometimes affect multiple genes; it also reports potential human-genome off-target sites for the gRNAs, including 76 for gRNA-175, with the highest-homology site showing 2.27±0.18% indel frequency.
  • Evident from the text: the study tests only two large DNA viruses, ADV and HSV1, and does not establish generalizability to all large viral genomes or in vivo vaccine/therapeutic applications.

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

Integrity signals

Registry

Crossref · 10.1371/journal.ppat.1004090

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 18 cited sources checked · 0 supported · 0 flagged

  • Findingreasoning

    The ratio of mutant to total viral genomes decreased dramatically from P1 to P2 during infectious viral particle packaging.

    “Furthermore, the ratio of mutant to total viral genomes decreased dramatically from P1 to P2 during infectious viral particle packaging.”
    Quote in the paperSubstantial claim
  • Findingnone given

    The more active Cas9 resulted in more mutations in the progeny virus contained.

    “Moreover, the more active Cas9 resulted in more mutations in the progeny virus contained.”
    Quote in the paperNot a substantial claim
  • Findingown data

    All three gRNAs (gRNA-173, gRNA-174, and gRNA-175) could induce site-specific mutations in the adenoviral genome.

    “All three gRNAs could induce site-specific mutations in the adenoviral genome (Figure 1C)”
    Quote in the paperSubstantial claim
  • Findingown data

    When the virus was substituted with ADV-DsRed, specific gRNA-175-induced RGN did not interfere with the viral replication process.

    “However, when the virus was substituted with ADV-DsRed harboring a red fluorescent protein from Discosoma coral (DsRed), specific gRNA-175-induced RGN did not interfere with the viral replication process (Figure 4D).”
    Quote in the paperSubstantial claim
  • Findingown data

    Deep sequencing of three highly homologous regions in RGN-treated (gRNA-175) P1 ADV-EGFP progeny viral genomes detected no off-target mutations.

    “Using RGN-treated (gRNA-175) P1 ADV-EGFP progeny viral genomes as the object, deep sequencing was performed on these three highly homologous regions, and no off-target mutations were detected (Table 1).”
    Quote in the paperSubstantial claim
  • May combine two claims. 40
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Findingown data

    Few of the viral genomes that resulted from double site-specific cleavages could form infectious viral particles, and the mutations in these genomes were rarely passed to the viral progeny.

    “However, few of the viral genomes that resulted from double site-specific cleavages could form infectious viral particles, and the mutations in these genomes were rarely passed to the viral progeny when progeny viruses were used to infect fresh cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    gRNA-175 was the most efficient (47.4%), followed by gRNA-174 (37.3%), whereas gRNA-173 was the least efficient (32.7%).

    “gRNA-175 was the most efficient (47.4%), followed by gRNA-174 (37.3%), whereas gRNA-173 was the least efficient (32.7%).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Mutant viral genome formation was the most efficient at an m.o.i. of between 1 and 10, and the efficiency of Cas9 cleavage and NHEJ repair decreased at a viral m.o.i. of 100.

    “Mutant viral genome formation was the most efficient at an m.o.i. of between 1 and 10, and the efficiency of Cas9 cleavage and NHEJ repair decreased at a viral m.o.i. of 100”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    In the few cells that were not transfected with CRISPR-Cas9 (approximately 5%), the wild-type virus replicated normally; therefore, the percentage of mutants decreased.

    “In the few cells that were not transfected with CRISPR-Cas9 (approximately 5%), the wild-type virus replicated normally; therefore, the percentage of mutants decreased.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    When the guide RNA was substituted with gRNA-174, the replication of ADV-EGFP was also inhibited; viral replication was further reduced when gRNA175 and gRNA174 were expressed.

    “When the guide RNA was substituted with gRNA-174, the replication of ADV-EGFP was also inhibited. Moreover, the viral replication was further reduced when gRNA175 and gRNA174 were expressed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Five of the eleven clones with indels had a GCG sequence missing, and three had a G missing.

    “Five of these eleven clones had a GCG sequence missing, and three had a G missing, which are both believed to be dominant mutations.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Co-expressing Cas9 with gRNA-174 and gRNA-175 induced simultaneous double site-specific cleavages within the viral genome, resulting in a DNA fragment approximately 100 bp shorter than the product of single site-specific cleavages.

    “When the viral genome was amplified by PCR, this double cleavage resulted in the formation of a DNA fragment that was approximately 100 bp shorter (Figure 3B) than the product of single site-specific cleavages by gRNA-174 or gRNA-175.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In RGN (gRNA-175)-expressing cells, the quantity of F1 was equal to that of the control, whereas F2 was significantly lower than that in the control.

    “In RGN (gRNA-175)-expressing cells, the quantity of F1 was equal to that of the control, whereas F2 was significantly lower than that in the control (Figure 4F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    At a viral m.o.i. of 0.1, the mutation efficiency declined, and no significant CPE was observed even after long incubation.

    “Surprisingly, at a viral m.o.i. of 0.1, the mutation efficiency declined, and at this relatively low m.o.i., no significant CPE was observed, even after a long incubation period post-CRISPR-Cas9 transfection (up to 6 days)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The mutation efficiency increased with the increase in Cas9-gRNA (pcw175) expression and reached its peak between 24 and 36 hours post-transfection.

    “The mutation efficiency increased with the increase in Cas9-gRNA (pcw175) expression and reached its peak between 24 and 36 hours post-transfection (Figure 4A)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus.

    “Furthermore, CRISPR-Cas9 can specifically inhibit the replication of the original virus, thereby significantly increasing the abundance of the recombinant virus among progeny virus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Viral genomes mutated by Cas9 cleavage and subsequent repair were detectable at 12 hours post-infection, and the percentage of mutant progeny genomes reached a maximum and was maintained at 47%-52% of the total genomes at 36 hours.

    “Viral genomes mutated by Cas9 cleavage and subsequent repair were detectable at 12 hours post-infection. As time progressed, the percentage of mutant progeny genomes reached a maximum and was maintained at 47%-52% of the total genomes at 36 hours (Figure 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In control cells, the viral logarithmic growth phase occurred at 24-36 hours post-infection, whereas in RGN (gRNA-175)-treated cells, the logarithmic growth phase was delayed to 36-48 hours, and the peak titer was 4-5-times lower than the viral titer of control cells.

    “In control cells, the viral logarithmic growth phase occurred at 24–36 hours post-infection, although in RGN (gRNA-175)-treated cells, the logarithmic growth phase was delayed to 36–48 hours, and the peak titer was 4–5-times lower than the viral titer of control cells (Figure 4D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nine of the twenty clones were wild type, and eleven clones had indels in the region complementary to gRNA-175.

    “Nine of the twenty clones were wild type (containing no mutations), and eleven clones had indels in the region complementary to the gRNA-175.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the eight mutants, the observed genotypes were: one missing a cytosine (D1), three with a guanine insertion (+1a), two with a cytosine insertion (+1b), one with an insertion of a 115-bp sequence (+115), and one mixture of two mutant viruses.

    “Of these eight mutants, one was missing a cytosine (D1), three harbored a guanine insertion (+1a), two contained a cytosine insertion (+1b), one had an insertion of a 115-bp sequence (+115, see Figure S4B for sequence), and one was a mixture of two mutant viruses (Figure S4C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The most similar homologous sequence to gRNA-206 in the HSV1 genome contained five mismatches and included a gap or six mismatches within the PAM sequence.

    “the most similar homologous sequence to gRNA-206 in the HSV1 genome contained five mismatches and included a gap or six mismatches within the PAM sequence (Table S6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A single HSV1-EGFP viral plaque was isolated from a 10−5 dilution and verified by PCR as pure virus with no wild-type HSV1 contamination.

    “A single HSV1-EGFP viral plaque was isolated from a 10−5 dilution and verified by PCR as pure virus with no wild-type HSV1 contamination (Figure 7G, 7H).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    All of the eight mutants included indels at the gRNA-206 site within the UL23 sequence.

    “all of which included indels at the gRNA-206 site within the UL23 sequence.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In cells treated with RGN and donor DNA, (5.8±1.7)×105 pfu/ml green plaques were obtained among (6.9±1.8)×106 pfu/ml total plaques, and the efficiency of homologous recombination increased to 8.41%.

    “In cells treated with RGN and donor DNA, (5.8±1.7)×105 pfu/ml green plaques were obtained among (6.9±1.8)×106 pfu/ml total plaques (Figure 7F), and the efficiency of homologous recombination increased to 8.41%.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 36 hours post infection, the titer for control viral progeny lacking RGN was 3.2×108 TCID50/ml, and the titer decreased to 6.31×107 TCID50/ml for viral progeny with RGN.

    “At 36 hours post infection, the titer for control viral progeny lacking RGN was 3.2×108 TCID50/ml, and the titer decreased to 6.31×107 TCID50/ml for the viral progeny with RGN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Progeny viruses obtained from the RGN system were single ADV-DsRed, confirmed by PCR and sequencing of an isolated red plaque.

    “A plaque was selected and examined after viral amplification by PCR and sequencing to further confirm that the progeny viruses were single ADV-DsRed (Figure 5C, 5D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the presence of RGN, ACV-resistant viral progeny reached titers of 3.16×107 TCID50/ml, which was 50.1% of the total virus.

    “in the presence of RGN, ACV-resistant viral progeny reached titers of 3.16×107 TCID50/ml (Figure 7C), which was 50.1% of the total virus.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Acyclovir is a known treatment for herpes infection and targets thymidine kinase.
    • Rests on an assumption: Drug-resistant, but not wild-type, viral strains can survive in the presence of 100 µg/ml ACV during titration.

  • May combine two claims.

    Findingown data

    The efficiency of homologous recombination increased to 2.6±0.57% with the RGN system, a significant increase compared with naturally occurring homologous recombination.

    “The efficiency of homologous recombination increased to 2.6±0.57% (Figure 5B), which was a significant increase compared with naturally occurring homologous recombination.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The off-target sites for gRNA-174, gRNA-173, and gRNA-206 present in the human genome are 123, 34, and 8, respectively.

    “The off-target sites for gRNA-174, gRNA-173, and gRNA-206 that are present in the human genome are 123, 34, and 8, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sequence alignment indicated that 76 gRNA-175 off-target sites are present in the human genome, of which 19 are located in exons of protein-coding genes.

    “Sequence alignment indicated that 76 gRNA-175 off-target sites are present in the human genome, in which 19 are located in the exons of protein-coding genes (Table S7).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    An appropriate amount of Cas9n:gRNA (1.5 μg pcw180 or pcw178) could induce homologous recombination with donor DNA in viral genomes when the concentration of Cas9n introduced into cells in the presence of donor DNA (pcw270) is controlled.

    “an appropriate amount of Cas9n:gRNA (1.5 µg pcw180 or pcw178) could induce homologous recombination with donor DNA in viral genomes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    DNA viral genomes can be repaired through NHEJ after site-specific cleavage by the CRISPR-Cas9 system, and the mutant genomes can be effectively transferred to progeny viruses.

    “Our data indicate that DNA viral genomes can be repaired through NHEJ after site-specific cleavage by the CRISPR-Cas9 system and that the mutant genomes can be effectively transferred to progeny viruses.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells, thereby promoting undesired off-target mutations.

    “as mentioned in several recent publications, Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells [12], [24], [25], [26], [27], thereby promoting undesired off-target mutations.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [12] L Cong, 2013, Multiplex genome engineering using CRISPR/Cas systems, Science source

      Not checked in the free run.

    • cites [24] Y Fu, 2013, High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells, Nat Biotechnol source

      Not checked in the free run.

    • cites [25] PD Hsu, 2013, DNA targeting specificity of RNA-guided Cas9 nucleases, Nat Biotechnol source

      Not checked in the free run.

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

    Premisecitation

    The CRISPR-Cas9 system requires the design of only a single guide sequence that matches the DNA targeted for cleavage.

    “The CRISPR-Cas9 system requires the design of only a single guide sequence that matches the DNA targeted for cleavage.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] M 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.

    Interpretationnone given

    The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the NHEJ and HDR pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency.

    “The CRISPR-Cas9 system robustly stimulates targeted double-stranded breaks in the genomes of DNA viruses, where the non-homologous end joining (NHEJ) and homology-directed repair (HDR) pathways can be exploited to introduce site-specific indels or insert heterologous genes with high frequency.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The genomes of viruses are much smaller than those of mammalian cells; therefore, cleavage of a DNA sequence in a virus is more likely to affect viral replication than mammalian cell replication.

    “The genomes of viruses are much smaller than those of mammalian cells; therefore, cleavage of a DNA sequence in a virus is more likely to affect viral replication than mammalian cell replication.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    CRISPR-Cas9-induced cellular genome repair efficiency is not a major concern for eukaryotic cells, but for viruses, it could be an issue.

    “CRISPR-Cas9-induced cellular genome repair efficiency is not a major concern for eukaryotic cells, but for viruses, it could be an issue.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The CRISPR-Cas9 system accurately controls the site of mutation and is a highly efficient strategy for the construction of large recombinant viral genomes.

    “The CRISPR-Cas9 system accurately controls the site of mutation and is a highly efficient strategy for the construction of large recombinant viral genomes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Compared with ZFNs and TALENs, the new CRISPR-Cas approach is more convenient and efficient.

    “Compared with well-known, site-specific genome editing technologies, such as ZFNs and TALENs, the new CRISPR-Cas approach is more convenient and efficient [10], [12].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [10] P Mali, 2013, RNA-guided human genome engineering via Cas9, Science source

      Not checked in the free run.

    • cites [12] L Cong, 2013, Multiplex genome engineering using CRISPR/Cas systems, Science source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    The highly effective, site-specific CRISPR-Cas9 genome editing system will promote more rapid development of large-genome viral vectors, attenuated vaccines against large DNA viruses, and an understanding of viral life cycles.

    “The highly effective, site-specific CRISPR-Cas9 genome editing system will promote more rapid development of large-genome viral vectors, attenuated vaccines against large DNA viruses, and an understanding of viral life cycles.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The cellular expression level of the RGN complex, viral m.o.i., and viral harvest time all affect the formation of mutant viral progeny.

    “Our results indicate that the cellular expression level of the RGN complex, viral m.o.i., and viral harvest time all affect the formation of mutant viral progeny.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Large quantities of viral genomes cannot be efficiently repaired after cleavage, which is likely the major reason for the inhibition of viral replication.

    “Our study demonstrates that large quantities of viral genomes cannot be efficiently repaired after cleavage, which is likely the major reason for the inhibition of viral replication.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Because progeny viruses are able to infect new cells after cell lysis, the effect of host-cell mutations on progeny viral replication is limited.

    “Because progeny viruses are able to infect new cells after cell lysis, the effect of host-cell mutations on progeny viral replication is limited.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Progeny viruses are able to infect new cells after cell lysis.

  • May combine two claims.

    Interpretationreasoning

    At 24 hours post-infection, more than 90% of the viral genomes were cleaved, and between 1% and 7% of the viral genomes were repaired by the host DNA repair machinery.

    “Considering that the percentage of indels was 19.2% among the total F2 genomes (Figure 4C), we predicted that at 24 hours post-infection, more than 90% of the viral genomes were cleaved, and between 1% and 7% of the viral genomes were repaired by the host DNA repair machinery.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Prior methods for HSV1 genome editing require amplification and purification to obtain viral genomic DNA, then co-transfection with homologous DNA sequences into cells followed by two-step counter-selection and multiple plaque isolation or insertion into a BAC system and several in vitro molecular biological procedures.

    “The genomic DNA is then co-transfected with homologous DNA sequences into cells followed by two-step counter-selection and multiple plaque isolation [30] or by inserting the viral genome into a BAC system and performing several in vitro molecular biological procedures [31].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [30] ES Mocarski, 1980, Molecular engineering of the herpes simplex virus genome: insertion of a second L-S junction into the genome causes additional genome inversions, Cell source

      Not checked in the free run.

    • cites [31] PK Agarwalla, 2012, Oncolytic herpes simplex virus engineering and preparation, Methods Mol Biol source

      Not checked in the free run.

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

    Study design and conductnone given

    Each experiment was performed in parallel, and triplicate samples were included in each reaction.

    “Each experiment was performed in parallel, and triplicate samples were included in each reaction.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Due to the presence of hundreds of viral genomes within a single infected cell, the limited efficiency of the DNA repair systems within cells may not be able to repair the fragmented viral genomes in time.

    “Alternatively, due to the presence of hundreds of viral genomes within a single infected cell, the limited efficiency of the DNA repair systems within cells may not be able to repair the fragmented viral genomes in time.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: There are hundreds of viral genomes within a single infected cell.
    • Rests on an assumption: The DNA repair systems within cells have limited efficiency.

  • May combine two claims.

    Premisereasoning

    Both prior processes require several weeks and are extremely laborious and time-consuming.

    “Both processes require several weeks and are extremely laborious and time-consuming.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Mean indel mutation frequencies were determined using deep sequencing (N = 3).

    “Mean indel mutation frequencies were determined using deep sequencing (N = 3).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Because viral genomes are much smaller than cellular genomes, which are restricted to the nucleus, once the relatively dissociated viral genome is cleaved by a nuclease, gene breakage and separation may be more likely to occur.

    “Because viral genomes are much smaller than cellular genomes, which are restricted to the nucleus, once the relatively dissociated viral genome is cleaved by a nuclease, gene breakage and separation may be more likely to occur.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Viral genomes are much smaller than cellular genomes, which are restricted to the nucleus.

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

    Interpretationreasoning

    Due to the compact structure of the viral genome, gene overlapping occurs; therefore, multiple genes might sometimes be affected by the single cleavage of a viral genome.

    “However, it is noteworthy that, due to the compact structure of the viral genome, gene overlapping occurs; therefore, multiple genes might sometimes be affected by the single cleavage of a viral genome.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationcitation

    The mutant virus formed in P1 cannot be passed to the P2 virus in equal proportions, which illustrates steric hindrance caused by the association of the Cas9 protein with specific DNA sequences, and the time that elapses during cleavage and repair may affect viral genome packaging.

    “Moreover, the mutant virus that is formed in P1 cannot be passed to the P2 virus in equal proportions, which illustrates the steric hindrance caused by the association of the Cas9 protein with specific DNA sequences [34], and the time that elapses during cleavage and repair may affect viral genome packaging.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] LS Qi, 2013, Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression, Cell source

      Not checked in the free run.

47 of 47

  • The paper relies on this without support.

    Large viral genomes (including Adenoviridae, Herpesviridae, and Poxviridae) lack readily available monoclonal restriction enzyme digestion sites, making enzyme digestion and ligation processes more difficult.

    “However, for those viruses with large viral genomes, including Adenoviridae, Herpesviridae, and Poxviridae, which have genome sizes larger than 30 Kb, monoclonal restriction enzyme digestion sites are not readily available, making the enzyme digestion and ligation processes more difficult.”

  • The paper relies on this without support.

    Use of the CRISPR-Cas9 system to induce mutations in non-integrating viral genomes has not been reported.

    “However, use of the CRISPR-Cas9 system to induce mutations in non-integrating viral genomes has not been reported, likely due to the genomic differences between viruses and cells.”

  • The paper relies on this without support.

    The genomes of DNA viruses are in a dissociated form, so DSBs may terminate viral replication.

    “Furthermore, unlike mammalian cells [33], the genomes of DNA viruses are in a dissociated form, so DSBs may terminate viral replication.”

  • The paper relies on this without support.

    The use of the CRISPR-Cas system for editing a non-integrating viral genome has not been previously reported.

    “To the best of our knowledge, the use of the CRISPR-Cas system for editing a non-integrating viral genome has not been previously reported.”

  • The paper relies on this without support.

    Acyclovir is a known treatment for herpes infection and targets thymidine kinase.

    “A known treatment for herpes infection is acyclovir (ACV), a drug that targets TK.”

  • The paper relies on this without support.

    Drug-resistant, but not wild-type, viral strains can survive in the presence of 100 µg/ml ACV during titration.

    “Drug-resistant, but not wild-type, viral strains can survive in the presence of 100 µg/ml ACV during titration.”

  • The paper relies on this without support.

    Progeny viruses are able to infect new cells after cell lysis.

    “Because progeny viruses are able to infect new cells after cell lysis, the effect of host-cell mutations on progeny viral replication is limited.”

  • The paper relies on this without support.

    Viral genomes are much smaller than cellular genomes, which are restricted to the nucleus.

    “Because viral genomes are much smaller than cellular genomes, which are restricted to the nucleus, once the relatively dissociated viral genome is cleaved by a nuclease, gene breakage and separation may be more likely to occur.”

  • The paper relies on this without support.

    There are hundreds of viral genomes within a single infected cell.

    “Alternatively, due to the presence of hundreds of viral genomes within a single infected cell, the limited efficiency of the DNA repair systems within cells may not be able to repair the fragmented viral genomes in time.”

  • The paper relies on this without support.

    The DNA repair systems within cells have limited efficiency.

    “Alternatively, due to the presence of hundreds of viral genomes within a single infected cell, the limited efficiency of the DNA repair systems within cells may not be able to repair the fragmented viral genomes in time.”

  • The paper relies on this without support.

    Mutant viruses possess replication priority under the cleavage pressure of the CRISPR-Cas9 system.

    “However, mutant viruses possess replication priority under the cleavage pressure of the CRISPR-Cas9 system.”

  • The paper relies on this without support.

    The virus has a short replication period and high progeny productivity.

    “Nevertheless, due to the short replication period and high progeny productivity of the virus, several rounds of CRISPR-Cas9 selection pressure could gradually increase the proportion of recombinant virus among total virus.”
12 of 12

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?

  • 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”

    From The CRISPR/Cas9 system facilitates clearance of the intrahepatic HBV templates in vivo (2014) · cites this paper as [35]

The citation lists haven’t been fetched yet

References 35 · Cited by 148

Research works printed but not linked: 1

  1. ZX Wu, 2013, Titration of recombinant adenovirus in liver cell line by enhanced green fluorescent protein reporter virus, Journal of Yunnan University, S1, 43

Authors
Bi, Y., Sun, L., Gao, D., Ding, C., Li, Z., Li, Y., Cun, W., & Li, Q.
Affiliations
Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
Corresponding author
  • Cun, Wei
  • Li, Qihan
ORCID
Venue
PLoS Pathogens
Publisher
Public Library of Science (PLoS)
Published
2014-05-01
Volume
10
Issue
5
Pages
e1004090
ISSN
  • 1553-7366 (ISSN-L)
  • 1553-7374
DOI
10.1371/journal.ppat.1004090
PMC
PMC4006927
OpenAlex
W1970966858
Semantic Scholar
998d280f3abef2083649c74c7cd66cb2e5cb832c
Cited by
148
References
35
Citations by year
2026: 3 · 2025: 5 · 2024: 5 · 2023: 5 · 2022: 17 · 2021: 13 · 2020: 15 · 2019: 7 · 2018: 19 · 2017: 14 · 2016: 25 · 2015: 14 · 2014: 6
FWCI
8.06
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS Pathogens (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
  • Broad Institute
  • Natural Science Foundation of Yunnan Province (2013FA053)
  • National Key Research and Development Program of China (2012CB518901)
  • Fundamental Research Funds for the Central Universities
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
CRISPR/Cas9, targeted genome editing, DNA viruses, adenovirus, herpes simplex virus type 1, attenuated vaccine strain, non-homologous end joining, homology-directed repair, indels, gene therapy vectors, viral replication inhibition
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Adenoviridae, Adenoviridae Infections, Amino Acid Sequence, Animals, Base Sequence, Cells, Cultured, Chlorocebus aethiops, Genetic Engineering, Humans, Molecular Sequence Data, Ribonucleases, Vero Cells, Viral Vaccines, Genome, Viral, RNA Editing, HEK293 Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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