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Cited by 220CRISPR and Genetic EngineeringVirus-based gene therapy researchViral-associated cancers and disorders

RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

Open access
Wang, Jianbin, Quake, Stephen R.·2014·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1410785111PMC PMC4246930
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Abstract

Significance: Latent viral infection is a major obstacle for effective antiviral treatment and presents a continuous risk to the host. The dormant viral genome during latent infection provides few therapeutic targets other than itself for antiviral drug development. This study demonstrates the clearance of latent Epstein–Barr virus genomes in a subpopulation of Burkitt’s lymphoma patient-derived cells with clustered regularly interspaced short palindromic repeat/Cas9 nuclease. Viral genome destruction leads to proliferation arrest and apoptosis in Epstein–Barr virus-infected cells, with no observed cytotoxicity to noninfected cells. Although many hurdles remain before this approach could be used in the clinic, this strategy may lead to a generalized approach to cure latent viral infections.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper shows that CRISPR/Cas9 can be targeted to the latent Epstein-Barr virus genome in Burkitt’s lymphoma cells, causing large viral deletions, proliferation arrest, apoptosis, and complete EBV clearance in a subpopulation of cells. It proposes RNA-guided endonuclease therapy as a potential strategy to cure latent herpesviridae infection, while noting that delivery and clinical development remain unresolved.

Key findings

  • In Raji cells, adding the EBV origin of replication (*oriP*) to the CRISPR plasmid increased transfection efficiency from <10% at 48 h to >60%.
  • Untreated Raji cells averaged 42 EBV genome copies per cell; treatment with the guide RNA mixture sgEBV1–7 reduced EBV load by ∼65% in live cells and ∼85% in dead cells. Single-cell analysis found that among 71 quality-controlled treated cells, 19 had no detectable EBV, 30 had dramatically decreased EBV load, and 22 were unchanged; the paper states that around 25% of treated cells were completely cleared of EBV and another 50% showed substantial decrease.
  • Guide RNAs targeting structural repeat regions (sgEBV1/2/6) completely stopped cell proliferation despite only a 26% decrease in EBV load, whereas sgEBV4/5 reduced the EBV genome by 85% but did not suppress proliferation as effectively as the full mixture.
  • Treatment caused apoptosis markers and no measurable proliferation change in EBV-negative DG-75 Burkitt’s lymphoma cells or primary human IMR90 lung fibroblasts, suggesting negligible cytotoxicity.

Methods

  • Burkitt’s lymphoma cell lines Raji, Namalwa, and DG-75, plus primary human lung fibroblast IMR-90, were cultured under standard conditions; CRISPR/Cas9 plasmids were modified from pX458 to include the pmax promoter, sgRNA, and EBV *oriP*, and delivered by nucleofection.
  • Seven guide RNAs were designed against the EBV B95-8 reference genome, targeting structural repeat regions, EBNA1, and EBNA3C/LMP1; editing was assessed by SURVEYOR assays, PCR, and Sanger sequencing.
  • Viral load was quantified by digital PCR on the Fluidigm BioMark system and by single-cell whole-genome amplification on the Fluidigm C1 system, with flow sorting and qPCR for EBV and human somatic loci.

Limitations

  • The paper states that further development into a practical therapeutic will require more sophisticated delivery approaches, which were beyond the scope of the study; it also states that off-target cleavage is a major concern for nuclease therapy, though it cites prior work suggesting off-target effects are not a substantial impediment for nonhuman genome targets.
  • The paper states that a single treatment did not eliminate all virus-infected cells, but argues that multiple doses could amplify efficacy geometrically and that partial elimination might allow immune control of residual latent infection.
  • Evident from the text: the work is limited to cultured cell lines and does not demonstrate in vivo delivery, animal-model efficacy, or direct off-target/safety assessment in this study; guide RNA delivery and plasmid replication are described as stochastic.

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

Integrity signals

Registry

Crossref · 10.1073/pnas.1410785111

Cites

37 of 37 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
    § RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

    Patient-derived cells from a Burkitt's lymphoma with latent Epstein-Barr virus infection showed dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome.

    “Patient-derived cells from a Burkitt's lymphoma with latent Epstein-Barr virus infection showed dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome.”
    Quote in the paperSubstantial claim
  • Findingown data

    After sgEBV2 transfection, PCR amplification of the whole repeat region produced an ~0.4-kb band at 5 or 7 days, representing an ~1.4-kb deletion expected from repair ligation between cuts in the first and last repeat unit.

    “After 5 or 7 d of sgEBV2 transfection, we obtained ∼0.4-kb bands from the same PCR amplification (Fig. 2B).The ∼1.4-kb deletion is the expected product of repair ligation between cuts in the first and the last repeat unit (Fig. 2A).”
    Quote in the paperSubstantial claim
  • Findingown data

    After sgEBV1-7 treatment, flow-cytometry scattering signals revealed altered cell morphology, with most cells shrinking and increasing granulation (P4 to P3 shift).

    “Flow-cytometry scattering signals clearly revealed alterations in the cell morphology after sgEBV1-7 treatment, as the majority of the cells shrank in size with increasing granulation (Fig. 3 B-D) (population P4 to P3 shift).”
    Quote in the paperSubstantial claim
  • Findingown data

    Annexin V staining revealed a distinct subpopulation of cells with intact cell membrane but exposed phosphatidylserine, suggesting cell death through apoptosis.

    “Annexin V staining revealed a distinct subpopulation of cells with intact cell membrane but exposed phosphatidylserine, suggesting cell death through apoptosis (Fig. 3 E-G).”
    Quote in the paperSubstantial claim
  • Findingown data

    Cells treated with a Cas9 plasmid with oriP but no sgEBV had an EBV load reduced by ~50%.

    “Cells treated with a Cas9-plasmid with oriP but no sgEBV had an EBV load that was reduced by ∼50%.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 23
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Findingown data

    Cells treated with Cas9 plasmids lacking oriP or sgEBV lost EGFP expression within a few days and proliferated at a rate similar to untreated controls.

    “As expected, cells treated with Cas9 plasmids that lacked oriP or sgEBV lost EGFP expression within a few days and proliferated with a rate similar rate to the untreated control group (Fig. 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transfection of a guide RNA mixture completely suppressed virally induced cell proliferation and restored the apoptosis pathway in the cells.

    “Transfection of a guide RNA mixture completely suppressed virally induced cell proliferation and restored the apoptosis pathway in the cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    sgEBV4/5 reduced the EBV genome by 85% but could not suppress cell proliferation as effectively as the full guide RNA mixture.

    “Although sgEBV4/5 reduced the EBV genome by 85%, they could not suppress cell proliferation as effectively as the full mixture (Fig. 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Around 25% of treated cells were completely cleared of EBV with no detectable viral DNA, and another 50% showed substantial EBV load decrease relative to untreated.

    “Around 25% of the treated cells were completely cleared of EBV with no detectable viral DNA, and another 50% of cells showed substantial EBV load decrease relative to the untreated sample.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Addition of the guide RNA mixture sgEBV1-7 further reduced EBV load, with live and dead cells showing ~65% and 85% decreases, respectively, compared with untreated controls.

    “The addition of the guide RNA mixture sgEBV1-7 to the transfection further reduced the EBV load.The live and dead cells have ∼65% EBV and 85% decreases, respectively, compared with the untreated control, and as previously discussed these cells did not proliferate.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For sgEBV1-7 treated cells, 71 single-cell products passed quality control and the EBV load distribution was dramatically wider; 22 cells had the same EBV load as untreated, 19 cells had no detectable EBV, and 30 cells displayed dramatic EBV load decrease.

    “For the sgEBV1-7 treated sample, 71 single-cell products passed the quality control and the EBV load distribution was dramatically wider (Fig. 4E).Although 22 cells had the same EBV load as the untreated cells, 19 cells had no detectable EBV and the remaining 30 cells displayed dramatic EBV load decrease from the untreated sample.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    CRISPR/Cas9 can delete regions between unique targets in the EBV genome, as shown by shorter PCR amplicons and Sanger sequencing after sgEBV4/5 and sgEBV3/5 treatment.

    “We further demonstrated that it is possible to delete regions between unique targets (Fig. 2C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with sgEBV1-7 did not change proliferation rates of EBV-negative Burkitt's lymphoma DG-75 cells or primary human lung fibroblast IMR90 cells at 8 and 9 days after transfection, suggesting negligible CRISPR cytotoxicity.

    “Eight and 9 d after transfection, the cell proliferation rates did not change from the untreated control groups, suggesting negligible cytotoxicity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Guide RNAs targeting structural sequences (sgEBV1/2/6) could stop cell proliferation completely, despite not eliminating the full EBV load (26% decrease).

    “Guide RNAs targeting the structural sequences (sgEBV1/2/6) could stop cell proliferation completely, despite not eliminating the full EBV load (26% decrease).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with the guide RNA mixture sgEBV1-7 resulted in no measurable cell proliferation and total cell count either remained constant or decreased.

    “Treatment with the mixed mixture sgEBV1-7 resulted in no measurable cell proliferation and the total cell count either remained constant or decreased (Fig. 3A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sixty-nine untreated single-cell products passed quality control and displayed a log-normal distribution of EBV load, with almost every cell displaying significant amounts of EBV genomic DNA.

    “Sixty-nine untreated single-cell products passed the quality control and displayed a log-normal distribution of EBV load (Fig. 4D), with almost every cell displaying significant amounts of EBV genomic DNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Bright-field microscopy showed apoptotic cell morphology and fluorescent staining demonstrated DNA fragmentation after sgEBV1-7 treatment.

    “Bright-field microscopy showed obvious apoptotic cell morphology (Fig. 3 H and I) and fluorescent staining demonstrated drastic DNA fragmentation (Fig. 3 J-M).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Plasmids with Cas9-oriP and a scrambled guide RNA maintained EGFP expression after 8 days but did not reduce the cell proliferation rate.

    “Plasmids with Cas9-oriP and a scrambled guide RNA maintained EGFP expression after 8 d, but did not reduce the cell proliferation rate.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

    DNA sequence recognition by CRISPR/Cas9 requires only a single 20-nt guide RNA and a protospacer adjacent motif, enabling rapid engineering and testing of many DNA cleavage sites.

    “DNA sequence recognition requires only a single 20-nt guide RNA and a protospacer adjacent motif, which enables one to rapidly engineer and test a large number of DNA cleavage sites (17)(18)(19)(20).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [17] source

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

    Premisecitation
    § RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

    It has been recognized that targeting latent infections with viral genome-specific nucleases would be useful, but engineering sequence-specific nucleases has hampered progress.

    “It has been recognized that it would be useful to target latent infections with viral genome-specific nucleases (3-5), but the challenges of engineering sequence-specific nucleases have hampered progress.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3]

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

    Premisecitation
    § RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection

    CRISPR/Cas9 is a naturally occurring bacterial immune system that protects bacteria from phage infection and has been harnessed for genome-engineering applications.

    “Clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 is a naturally occurring bacterial immune system that uses a novel nuclease system to protect bacteria from phage infection (6-9), and it has recently been harnessed for a variety of genome-engineering applications (10)(11)(12)(13)(14)(15)(16)(17).”
    Quote in the paperSubstantial claim0 of 10 sources checked
    • cites [6]

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

    Premisecitation

    The Raji cell line was the first established long-term culture from Burkitt's lymphoma patients and is one of the most extensively studied EBV models.

    “The Raji cell line was the first established long-term culture from Burkitt's lymphoma patients (22,23), and is one of the most extensively studied EBV models.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22]

      Not checked in the free run.

    • cites [23]

      Not checked in the free run.

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

    Interpretationreasoning

    The plasmid transfection and replication process is likely quite stochastic, so some cells will receive different subsets or mixtures of the guide RNA mixture, which might affect treatment efficiency.

    “Although we provided seven guide RNAs at the same molar ratio, the plasmid transfection and replication process is likely quite stochastic.Some cells will inevitably receive different subsets or mixtures of the guide RNA mixture, which might affect the treatment efficiency.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Prior studies of endonuclease treatment of herpesviridae infections had significant limitations: meganuclease treatment of cell lines prevented HSV infection but did not explore latent infections, and another latency treatment used fibroblasts rather than neurons and did not show complete virus elimination.

    “Prior studies of the efficacy of endonuclease treatment of herpesviridae infections, however, had significant limitations.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [28]

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    • cites [5]

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  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Single-cell whole-genome amplification products were quality-controlled by four-loci human somatic DNA quantitative PCR, requiring positive amplification from at least one locus.

    “For quality-control purposes, we performed four-loci human somatic DNA quantitative PCR on each single-cell amplification product (27) and required positive amplification from at least one locus.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Suppressing EBV activity may restore the apoptosis process, which could explain the cell death observed after EBV genome destruction.

    “Suppressing EBV activity may therefore restore the apoptosis process, which could explain the cell death observed in our experiment.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Previous studies attributed the tumorigenic ability of EBV to its interruption of host-cell apoptosis.

    “Previous studies have attributed the tumorigenic ability of EBV to its interruption of host-cell apoptosis (26).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26]

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Because a practical therapeutic could be administered in multiple doses, complete elimination of all virus-infected cells with a single treatment is not necessary; the geometric reduction effect from multiple doses will amplify efficacy.

    “Because any practical therapeutic could be administered in multiple doses, it is not necessary to obtain complete elimination of all virus-infected cells with a single treatment; the geometric reduction effect from multiple doses will amplify the efficacy of any such treatment.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Off-target cleavage is a major concern for any nuclease therapy, but studies show off-target effects are not a substantial impediment to human genome-engineering applications and do not appear to be an obstacle for applications targeting nonhuman genomes.

    “Together with recent improvements on CRISPR target specificity with dual-guide RNA designs (33)(34)(35), these studies show that off-target effects are not a substantial impediment to human genome-engineering applications and certainly do not appear to be an obstacle for applications that target nonhuman genomes (36).”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [33] source

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

    Premisenone given

    Continuous Burkitt's lymphoma cell lines derived from different patients provide a convenient and naturally occurring renewable source of material for EBV studies, in contrast to the lack of easily available HSV models.

    “The continuous Burkitt's lymphoma cell lines derived from different patients provide a convenient and naturally occurring renewable source of material for EBV studies, which is in contrast to the lack of easily available HSV models.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The lack of an adequate in vitro model for HSV infection has hampered antiviral research.
25 of 25

  • The paper relies on this without support.

    Existing antiviral therapies cannot effectively eradicate latent infection, so a cure is lacking for many prevalent viral diseases.

    “Although standard antiviral therapies can suppress active viral replication, no existing treatment can effectively eradicate latent infection and therefore a cure is lacking for many prevalent viral diseases.”

  • The paper relies on this without support.

    The dormant viral genome during latent infection provides few therapeutic targets other than itself for antiviral drug development.

    “The dormant viral genome during latent infection provides few therapeutic targets other than itself for antiviral drug development.”

  • The paper relies on this without support.

    EBNA1 is crucial for many EBV functions, including gene regulation and latent genome replication.

    “EBNA1 is crucial for many EBV functions, including gene regulation and latent genome replication.”

  • The paper relies on this without support.

    EBNA3C and latent membrane protein-1 are essential for host cell transformation.

    “EBNA3C and latent membrane protein-1 are essential for host cell transformation, and we designed guide RNAs sgEBV3 and sgEBV7 to target the 5′ exons of these two proteins, respectively.”

  • The paper relies on this without support.

    The EBV genome encodes about 85 genes, several of which are essential for lytic or latent infection.

    “The EBV genome encodes about 85 genes, several of which are essential for lytic or latent infection.”

  • The paper relies on this without support.

    EBNA1 binds to the EBV origin of replication (oriP) to maintain viral episomes and also regulates expression of other viral genes.

    “EBNA1 binds to the EBV origin of replication (oriP) to maintain viral episomes; it also regulates expression of other viral genes.”

  • The paper relies on this without support.

    The lack of an adequate in vitro model for HSV infection has hampered antiviral research.

    “Thus far, the lack of an adequate in vitro model for HSV infection has hampered antiviral research.”
7 of 7

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?

  • In a Burkitt's lymphoma cell line with latent EBV infection, CRISPR-Cas9 treatment inhibited cell proliferation and reduced viral load.

    “Similarly, in a Burkitt's lymphoma cell line with latent EBV infection, CRISPR-Cas9 treatment inhibited cell proliferation and reduced viral load.42”

    From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [42]

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Related

Authors
Wang, J., & Quake, S. R.
Affiliations
  • Department of Bioengineering,
  • Howard Hughes Medical Institute (US)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2014-08-25
Volume
111
Issue
36
Pages
13157-13162
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1410785111
PMC
PMC4246930
OpenAlex
W2084747588
Cited by
220
References
37
Citations by year
2026: 7 · 2025: 11 · 2024: 7 · 2023: 9 · 2022: 18 · 2021: 27 · 2020: 15 · 2019: 24 · 2018: 27 · 2017: 22 · 2016: 28 · 2015: 22 · 2014: 3
FWCI
10.24
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (U01HL099995, U01HL099999)
  • National Cancer Institute (P01CA139490, U54CA151459)
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
  • Health Sciences › Medicine › Oncology › Viral-associated cancers and disorders
Keywords
CRISPR/Cas9, RNA-guided endonuclease, viral latency, herpesvirus, Epstein-Barr virus, Burkitt lymphoma, antiviral gene therapy, viral load reduction
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Base Sequence, Endonucleases, Herpesvirus 4, Human, Humans, Models, Biological, Molecular Sequence Data, Genome, Viral, Sequence Deletion, RNA Editing, Epstein-Barr Virus Infections, Cell Line, Tumor, Cell Proliferation, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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