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Cited by 527CRISPR and Genetic EngineeringHIV Research and TreatmentVirus-based gene therapy research

RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

Open access
Hu, Wenhui, Kaminski, Rafal, Yang, Fan, Zhang, Yonggang, Cosentino, Laura, Li, Fang, Luo, Biao, Alvarez-Carbonell, David +4 more·2014·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.1405186111PMC PMC4128125
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Abstract

Significance: For more than three decades since the discovery of HIV-1, AIDS remains a major public health problem affecting greater than 35.3 million people worldwide. Current antiretroviral therapy has failed to eradicate HIV-1, partly due to the persistence of viral reservoirs. RNA-guided HIV-1 genome cleavage by the Cas9 technology has shown promising efficacy in disrupting the HIV-1 genome in latently infected cells, suppressing viral gene expression and replication, and immunizing uninfected cells against HIV-1 infection. These properties may provide a viable path toward a permanent cure for AIDS, and provide a means to vaccinate against other pathogenic viruses. Given the ease and rapidity of Cas9/guide RNA development, personalized therapies for individual patients with HIV-1 variants can be developed instantly.

AI snapshot

Written from the paper’s full text.

TL;DR

Cas9/guide RNA targeting the HIV-1 LTR U3 region erased integrated proviral DNA and blocked viral reactivation in latently infected microglial, promonocytic, and T cell models. Stable expression of multiplex LTR-A/B gRNAs also protected cells from new HIV-1 infection without detected off-target editing.

Key findings

  • Cas9/gRNAs targeting LTRs A–D reduced TSA-induced EGFP reactivation in latently infected CHME5 microglial cells, suppressed LTR-driven luciferase in TZM-bI cells, and produced indels in the LTR U3 region.
  • Multiplex LTR-A/B expression excised the entire 9,709-bp integrated HIV-1 genome spanning the 5′ to 3′ LTRs; in U1 cells, long-range PCR identified parental fragments of 9,935 bp and 10,176 bp and edited fragments of 833, 670, and 1,102 bp.
  • Stable Cas9/LTR-A/B-expressing TZM-bI clones failed to support new HIV-1 replication at 2 d postinfection with VSV-G-pseudotyped pNL4-3-ΔE-EGFP, and similar immunity was observed with native X4 pNL4-3 and R5 SF162/JRFL strains.
  • Whole-genome sequencing called 676,105 indels; among 92 potential off-target sites, only 8 overlapped called indels, with 6 in both samples and 2 only in the U6-CAG control, while two on-target HIV-1 LTR indels occurred only in the LTR-A/B subclone.

Methods

  • Humanized Cas9 vectors pX260 and pX330 were used to express single or multiplex gRNAs A–D targeting the HIV-1 LTR U3 region, avoiding conserved transcription factor binding sites.
  • Latently infected models included CHME5 microglial cells, U1 promonocytic cells, J-Lat T cells, and TZM-bI reporter cells; assays included EGFP flow cytometry, SURVEYOR nuclease assay, Sanger sequencing, PCR/TA cloning, GenomeWalker integration-site mapping, p24 ELISA, and luciferase reporter assays.
  • Off-target assessment used SURVEYOR/Sanger at predicted sites and whole-genome sequencing with GATK v.2.8.1 against hg19 and HIV-1 references at 15–30× coverage.

Limitations

  • The paper states that detectable residual viral load and reactivation may result from cell population heterogeneity and/or incomplete genome editing.
  • The paper states that whole-genome sequencing had limited 15–30× coverage, limited indel-calling confidence, possible false negatives, and substantial intersample indel-calling discrepancy likely due to limited confidence, coverage, and cellular heterogeneity.
  • Evident from the text: the work is in vitro in cell lines, with no animal-model or in vivo delivery data; the authors state that testing in animal models with stable Cas9/LTR-gRNA expression is an important next step.

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

Integrity signals

Registry

Crossref · 10.1073/pnas.1405186111

Cites

43 of 43 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 26 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    Ablation of HIV-1 genome by Cas9/LTR-A/B gRNAs was validated in latently infected J-Lat T cells harboring integrated HIV-R7/E-/EGFP using flow cytometry analysis, SURVEYOR assay, and PCR genotyping.

    “We also validated the ablation of HIV-1 genome by Cas9/ LTR-A/B gRNAs in latently infected J-Lat T cells harboring integrated HIV-R7/E-/EGFP (21) using flow cytometry analysis, SURVEYOR assay, and PCR genotyping”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] source

      Not checked in the free run.

  • Findingown data

    Single or multiplex gRNAs targeting 5′ and 3′ LTRs effectively eradicated the entire HIV-1 genome.

    “Single or multiplex gRNAs targeting 5′ and 3′ LTRs effectively eradicated the entire HIV-1 genome”
    Quote in the paperSubstantial claim
  • Findingown data

    GenomeWalker mapping detected two integrated proviral DNA copies at chromosomes Xp11-4 and 2p21 in U1 cells.

    “GenomeWalker mapping detected two integrated proviral DNA copies at chromosomes Xp11-4 (Fig. 2A) and 2p21 (Fig. S5A) in U1 cells”
    Quote in the paperSubstantial claim
  • Findingown data

    Expressing gRNAs plus Cas9 markedly decreased the fraction of TSA-induced EGFP-positive CHME5 cells.

    “Expressing of gRNAs plus Cas9 markedly decreased the fraction of TSA-induced EGFPpositive CHME5 cells”
    Quote in the paperSubstantial claim
  • Findingown data

    Indels for LTRs A-D were detected using a Cel I nuclease-based heteroduplex-specific SURVEYOR assay.

    “We detected insertion/ deletion gene mutations (indels) for LTRs A-D (Fig. 1B and Fig. S2B) using a Cel I nuclease-based heteroduplex-specific SURVEYOR assay”
    Quote in the paperSubstantial claim
  • May combine two claims. 29
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 9

  • May combine two claims.

    Findingown data

    In U1 cells expressing LTR-A/B gRNAs and Cas9, two additional DNA fragments of 833 and 670 bp in chromosome X and one additional 1,102-bp fragment in chromosome 2 were found; thus gRNAs A/B enabled Cas9 to excise the HIV-1 5′-3′ LTR-spanning viral genome segment in both chromosomes.

    “In U1 cells expressing LTR-A/B gRNAs and Cas9, we found two additional DNA fragments of 833 and 670 bp in chromosome X and one additional 1,102-bp fragment in chromosome 2. Thus, gRNAs A/B enabled Cas9 to excise the HIV-1 5′-3′ LTR-spanning viral genome segment in both chromosomes”
    Quote in the paperSubstantial claim

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

    Findingown data

    Analysis of ±300 bp flanking each indel against LTR-A/-B-targeted sites and predicted/potential gRNA off-target sites identified only 8 overlapped regions of 92 potential off-target sites against 676,105 indels: 6 indels occurring in both samples and 2 only in the U6-CAG control.

    “we identified only 8 overlapped regions of 92 potential off-target sites against 676,105 indels: 6 indels occurring in both samples, and 2 only in the U6-CAG control”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Long-term expression of Cas9/LTR-A/B gRNAs did not adversely affect cell growth or viability, suggesting a low occurrence of off-target interference with the host genome or Cas9-induced toxicity in this model.

    “The long-term expression of Cas9/LTR-A/B gRNAs did not adversely affect cell growth or viability, suggesting a low occurrence of off-target interference with the host genome or Cas9-induced toxicity in this model”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Sequencing of PCR products revealed that the entire 5′-3′ LTR-spanning viral genome was deleted, yielding a 351-bp fragment via a 190-bp excision between cleavage sites A and B, and a 682-bp fragment with a 175-bp insertion and a 27-bp deletion at the LTR-A and -B sites.

    “sequencing of the PCR products revealed that the entire 5′-3′ LTR-spanning viral genome was deleted, yielding a 351-bp fragment via a 190-bp excision between cleavage sites A and B (Fig. 1H and Fig. S4), and a 682-bp fragment with a 175-bp in- sertion and a 27-bp deletion at the LTR-A and -B sites, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Long-range PCR identified a 9,935-bp DNA fragment representing the entire 9,709-bp proviral HIV-1 DNA plus a flanking 226-bp X-chromosome-derived sequence, and a 10,176-bp fragment containing the 9,709-bp HIV-1 genome plus its flanking 467-bp chromosome-2-derived sequence, in parental or empty-vector U1 cells.

    “A 9,935bp DNA fragment representing the entire 9,709-bp proviral HIV-1 DNA plus a flanking 226-bp X-chromosome-derived sequence (Fig. 2A), and a 10,176-bp fragment containing 9,709-bp HIV-1 genome plus its flanking 2-chromosome-derived 467 bp (Fig. S5 A and B) were identified by the long-range PCR analysis of the parental control or empty-vector (U6-CAG) U1 cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Progressively shorter gRNA segments yielded increasing off-target cleavage sites 100% matched to corresponding on-target sequences; NGG+13 bp yielded 6, 0, 2, and 9 off-target sites, whereas NGG+12 bp yielded 16, 5, 16, and 29.

    “progressively shorter gRNA segments yielded increasing off-target cleavage sites 100% matched to corresponding on-target sequences (i.e., NGG+13 bp yielded 6, 0, 2, and 9 off-target sites, respectively, whereas NGG+12 bp yielded 16, 5, 16, and 29)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Multiplex expression of LTR-A/B gRNAs in mixed clonal CHME5 cells caused deletion of a 190-bp fragment between A and B target sites and led to indels to various extents.

    “Multiplex expression of LTR-A/B gRNAs in mixed clonal CHME5 cells caused deletion of a 190-bp fragment between A and B target sites and led to indels to various extents”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cells stably expressing Cas9/LTR-A/B gRNAs failed to support HIV-1 replication at 2 days postinfection, indicating that they were immunized effectively against new HIV-1 infection.

    “Unlike the control U6-CAG cells, the cells stably expressing Cas9/LTR-A/B gRNAs failed to support HIV-1 replication at 2 d postinfection, indicating that they were immunized effectively against new HIV-1 infection”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    U1 cells expressing LTR-A/B gRNAs and Cas9 exhibited substantially decreased HIV-1 viral load by functional p24 ELISA replication assay and real-time PCR analysis.

    “These cells exhibited substantially decreased HIV-1 viral load, shown by the functional p24 ELISA replication assay (Fig. 2C) and real-time PCR analysis (Fig. S5 C and D)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Combined expression of LTR-C/D-targeting gRNAs in TZM-bI cells caused excision of the predicted 302-bp viral DNA sequence and emergence of the residual 194-bp fragment.

    “the combined expression of LTR-C/D-targeting gRNAs in these cells caused excision of the predicted 302-bp viral DNA sequence, and emergence of the residual 194-bp fragment”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The preexistence of the Cas9/gRNA system in cells leads to a rapid elimination of new HIV-1 before it integrates into the host genome.

    “Interestingly, the preexistence of the Cas9/gRNA system in cells leads to a rapid elimination of the new HIV-1 before it integrates into the host genome”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PCR genotyping of LTR-A/B subclones shows indels and intact or combined LTR.

    “(H) PCR genotyping of LTR-A/B subclones (#8, #13) using primers to amplify DNA fragment covering HIV-1 LTR U3/R/U5 regions (-411 to +129) shows indels (a, deletion; c, insertion) and “intact” or combined LTR (b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    LTR-A/B stable cells, but not control U6-CAG cells, are resistant to new infection by pNL4-3-ΔE-EGFP HIV-1 reporter virus.

    “(D) Representative phase-contrast/ fluorescence micrographs show that LTR-A/B stable but not control (U6-CAG) cells are resistant to new infection by pNL4-3-ΔE-EGFP HIV-1 reporter virus (green).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Subcloning of LTR-A/B stable clones reveals complete loss of reporter reactivation and elimination of pNL4-3-ΔGag-d2EGFP proviral genome.

    “(E-G) Subcloning of LTR-A/B stable clones reveals complete loss of reporter reactivation determined by EGFP flow cytometry (E) and elimination of pNL4-3-ΔGag-d2EGFP proviral genome detected by standard (F) and real-time (G) PCR amplification of genomic DNA for EGFP and HIV-1 Rev response element (RRE); β-actin is a DNA purification and loading control.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Two indel alleles were identified from 15 sequenced clonal amplicons.

    “(Lower) Two indel alleles identified from 15 sequenced clonal amplicons.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Immunocytochemistry and Western blot analyses confirm expression of Flag-Cas9 in TZM-bI stable clones selected with puromycin.

    “(A) Immunocytochemistry (ICC) and Western blot (WB) analyses with anti-Flag antibody confirm the expression of Flag-Cas9 in TZM-bI stable clones puromycin (1 μg/mL) selected for 2 wk.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    LTR-A/B gRNAs induce the indicated on-target indels but no off-target indels, consistent with prior findings using deep sequencing of PCR products covering predicted/potential off-target sites.

    “The results suggest that LTR-A/B gRNAs induce the indicated ontarget indels but no off-target indels, consistent with prior findings using deep sequencing of PCR products covering predicted/ potential off-target sites”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites (14,(24)(25)(26)(27)

      Not checked in the free run.

    • Rests on an assumption: The human genome contains numerous endogenous retroviral genomes potentially susceptible to HIV-1-directed gRNAs.

  • May combine two claims.

    Premisenone given

    Introduction of nucleases into cells via nuclease-based genomic editing approaches remains inefficient and partially selective for removing the entire HIV-1 genome.

    “However, introduction of nucleases into cells via these nuclease-based genomic editing approaches remains inefficient and partially selective to remove the entire HIV-1 genome”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Nuclease-based approaches have been used to disrupt HIV-1 entry coreceptors CCR5 or CXCR4 and proviral DNA-encoding viral proteins.

    “These approaches have been used to disrupt HIV-1 entry coreceptors C-C chemokine receptor 5 (CCR5) or C-C-C chemokine receptor 4 (CXCR4) and proviral DNA-encoding viral proteins”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [8] source

      Not checked in the free run.

    • cites [9] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Engineered nucleases including ZFN, TALEN, and CRISPR Cas9 can disrupt target genes and have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome.

    “Advances in the engineered nucleases including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) that can disrupt target genes have raised prospects of selectively deleting HIV-1 proviral DNA integrated into the host genome”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [7] source

      Not checked in the free run.

    • cites [8] source

      Not checked in the free run.

    • cites [9] source

      Not checked in the free run.

    • cites [10] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    LTR-targeting Cas9/gRNAs A/B eradicates the HIV-1 genome and blocks its reactivation in latently infected microglial cells.

    “These results indicate that LTR-targeting Cas9/gRNAs A/B eradicates the HIV-1 genome and blocks its reactivation in latently infected microglial cells”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The latently infected cell lines used (CHME5, U1, J-Lat, TZM-bI) adequately model human latent HIV-1 reservoir cells for assessing eradication and immunization.

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

    Interpretationreasoning

    The detectable but low residual viral load and reactivation may result from cell population heterogeneity and/or incomplete genome editing.

    “The detectable but low residual viral load and reactivation may result from cell population heterogeneity and/or incomplete genome editing”
    Quote in the paperSubstantial claim

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

    Premisecitation

    HIV-1 persists in approximately 10^6 cells per patient during HAART and is linked to comorbidities including heart and renal diseases, osteopenia, and neurological disorders.

    “HIV-1 persists in ∼10 6 cells per patient during HAART, and is linked to comorbidities including heart and renal diseases, osteopenia, and neurological disorders”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [5] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The multiplex LTR-gRNAs/Cas9 system efficiently suppresses HIV-1 replication and reactivation in latently HIV-1-infected reservoir (microglial, monocytic, and T) cells and in TZM-bI cells.

    “Taken together, our results suggest that the multiplex LTR-gRNAs/Cas9 system efficiently suppresses HIV-1 replication and reactivation in latently HIV-1-infected “reservoir” (microglial, monocytic, and T) cells typical of human latent HIV-1 infection, and in TZM-bI cells highly sensitive for detecting HIV-1 transcription and reactivation”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The latently infected cell lines used (CHME5, U1, J-Lat, TZM-bI) adequately model human latent HIV-1 reservoir cells for assessing eradication and immunization.

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

    Interpretationreasoning

    The residual HIV-1 genome may reflect the presence of trace Cas9/gRNA-negative cells.

    “The residual HIV-1 genome (Fig. 1 F-H) may reflect the presence of trace Cas9/gRNA-negative cells”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    CCR5 gene-targeting ZFNs are in phase II clinical trials for HIV-1/AIDS treatment.

    “CCR5 gene-targeting ZFNs are in phase II clinical trials for HIV-1/AIDS treatment”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [11] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The additional DNA fragments did not emerge via circular LTR integration because circular LTR viral genome configuration was absent in parental U1 cells and is short lived and intolerant to repeated passaging.

    “The additional fragments did not emerge via circular LTR integration, because it was absent in the parental U1 cells, and such circular LTR viral genome configuration occurs immediately after HIV-1 infection but is short lived and intolerant to repeated passaging”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    HAART profoundly reduces HIV-1 replication and plasma viremia but does not suppress low-level viral genome expression and replication in tissues or target the latently infected cells that serve as an HIV-1 reservoir.

    “HAART profoundly reduces viral replication in cells that support HIV-1 infection and reduces plasma viremia to a minimal level but neither suppresses low-level viral genome expression and replication in tissues nor targets the latently infected cells that serve as a reservoir for HIV-1”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [3] source

      Not checked in the free run.

    • cites [4] source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    U1 is a chronically HIV-1 infected promonocytic cell subclone that exhibits low-level constitutive viral gene expression and replication and is an HIV-1 latency model for infected perivascular macrophages and monocytes.

    “The promonocytic U-937 cell subclone U1, an HIV-1 latency model for infected perivascular macrophages and monocytes, is chronically HIV-1 infected and exhibits low-level constitutive viral gene expression and replication”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The substantial intersample indel-calling discrepancy suggests probable off-target effects but most likely results from limited confidence, limited WGS coverage (15-30×), and cellular heterogeneity.

    “Such substantial intersample indel-calling discrepancy suggests the probable off-target effects but most likely results from its limited confidence, limited WGS coverage (15-30×), and cellular heterogeneity”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    LTR-directed gRNA/Cas9 eradicates the HIV-1 genome and effectively immunizes target cells against HIV-1 reactivation and infection with high specificity and efficiency.

    “Here, we found that LTR-directed gRNA/ Cas9 eradicates the HIV-1 genome and effectively immunizes target cells against HIV-1 reactivation and infection with high specificity and efficiency”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The latently infected cell lines used (CHME5, U1, J-Lat, TZM-bI) adequately model human latent HIV-1 reservoir cells for assessing eradication and immunization.
    • Rests on an assumption: The HIV-1 LTR U3 target sites selected are conserved across clinically relevant HIV-1 variants.

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

    Premisecitation

    TALEN and Cas9 have been tested experimentally for efficient disruption of CCR5 and CXCR4.

    “Similarly, TALEN and Cas9 have been tested experimentally for efficient disruption of CCR5 and CXCR4”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites (9,28,(34)(35)(36)(37)

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    These properties may provide a viable path toward a permanent or sterile HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses.

    “These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure, and perhaps provide a means to eradicate and vaccinate against other pathogenic viruses”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Cas9/gRNA can be delivered to and expressed in latently infected reservoir cells in patients sufficiently for therapeutic effect.

  • May combine two claims.

    Interpretationreasoning

    The combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus.

    “Our combined approaches minimized off-target effects while achieving high efficiency and complete ablation of the genomically integrated HIV-1 provirus”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Functional knockout of CCR5 in autologous CD4 T cells of a small cohort revealed that in one out of four enrolled subjects, the viral load remained undetectable at the time of treatment.

    “Functional knockout of CCR5 in autologous CD4 T cells of a small cohort of patients revealed that in one out of four enrolled subjects, the viral load remained undetectable at the time of treatment”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] source

      Not checked in the free run.

35 of 35

  • The paper relies on this without support.

    The latently infected cell lines used (CHME5, U1, J-Lat, TZM-bI) adequately model human latent HIV-1 reservoir cells for assessing eradication and immunization.

    “inactivating viral gene expression and replication in latently infected microglial, promonocytic, and T cells”

  • The paper relies on this without support.

    The HIV-1 LTR U3 target sites selected are conserved across clinically relevant HIV-1 variants.

    “We identified highly specific targets within the HIV-1 LTR U3 region”

  • The paper relies on this without support.

    Cas9/gRNA can be delivered to and expressed in latently infected reservoir cells in patients sufficiently for therapeutic effect.

    “These properties may provide a viable path toward a permanent or “sterile” HIV-1 cure”

  • The paper relies on this without support.

    The human genome contains numerous endogenous retroviral genomes potentially susceptible to HIV-1-directed gRNAs.

    “the human genome contains numerous endogenous retroviral genomes that are potentially susceptible to HIV-1directed gRNAs”

  • The paper relies on this without support.

    HIV-1 LTR sequences targeted by the gRNAs are sufficiently conserved across HIV-1 strains for the system to be independent of strain diversity.

    “The preventative vaccination is independent of HIV-1 strain's diversity because the system targets genomic sequences regardless of how the viruses enter the infected cells.”

  • The paper relies on this without support.

    The cell line models used (CHME5, U1, TZM-bI) are representative of latently infected patient cells for testing HIV-1 eradication.

    “Our results show that the HIV-1 Cas9/ gRNA system has the ability to target more than one copy of the LTR, which are positioned on different chromosomes, suggesting that this genome-editing system can alter the DNA sequence of HIV-1 in latently infected patient's cells harboring multiple proviral DNAs.”
6 of 6

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?

  • Another independent study harnessed CRISPR-Cas9 to mutate the HIV-1 LTR U3 region and gained similar results.

    “Recently, another independent study harnessed CRISPR-Cas9 to mutate HIV-1 LTR U3 region and gained similar results.27”

    From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [27]

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. ; UNAIDS Global Report: UNAIDS Report on the Global AIDS Epidemic 2012 (Joint United Nations Programme on HIV/AIDS, Geneva, 2012).

Cited by

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Related

Authors
Hu, W., Kaminski, R., Yang, F., Zhang, Y., Cosentino, L., Li, F., Luo, B., Alvarez-Carbonell, D., Garcia-Mesa, Y., Karn, J., Mo, X., & Khalili, K.
Affiliations
  • Temple University (US)
  • Fox Chase Cancer Center (US)
  • Case Western Reserve University (US)
  • Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106; and
  • Sichuan University (CN)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2014-07-21
Volume
111
Issue
31
Pages
11461-11466
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.1405186111
PMC
PMC4128125
OpenAlex
W1978917197
Semantic Scholar
fcee539d4cff89fc706ae07c1b2ae4dd88f32ea8
Cited by
527
References
43
Citations by year
2026: 12 · 2025: 18 · 2024: 20 · 2023: 26 · 2022: 33 · 2021: 47 · 2020: 49 · 2019: 47 · 2018: 45 · 2017: 60 · 2016: 89 · 2015: 75 · 2014: 6
FWCI
31.46
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 Institute of Mental Health (R25MH080661, R01MH093271, P30MH092177)
  • National Institute of Allergy and Infectious Diseases (U19AI096113)
  • National Institute of Neurological Disorders and Stroke (R01NS087971)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Immunology and Microbiology › Virology › HIV Research and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
HIV-1, CRISPR/Cas9, HIV latency, viral reactivation, antiretroviral therapy, microglia, T cells, off-target effects
MeSH terms
Base Sequence, Cell Line, Humans, Molecular Sequence Data, RNA, Vaccination, HIV-1, HIV Infections, Genome, Human, HIV Long Terminal Repeat, RNA Editing, Virus Latency, HEK293 Cells, RNA, Small Untranslated, Clustered Regularly Interspaced Short Palindromic Repeats

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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.

What counts as a claim ›How we measure confidence ›How we check this ›

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