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Cited by 19CRISPR and Genetic EngineeringRetinal Development and DisordersRNA Interference and Gene Delivery

Characterization of RNA editing and gene therapy with a compact CRISPR-Cas13 in the retina

Open access
Kumar, Satheesh, Hsiao, Yi-Wen, Wong, Vickie H. Y., Aubin, Deborah, Wang, Jiang-Hui, Lisowski, Leszek, Rakoczy, Elizabeth P., Li, Fan +4 more·2024·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2408345121PMC PMC11551378

Published version of a preprint

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Abstract

CRISPR-Cas13 nucleases are programmable RNA-targeting effectors that can silence gene expression in a transient manner. Recent iterations of Cas13 nucleases are compact for adeno-associated virus (AAV) delivery to achieve strong and persistent expression of various organs in a safe manner. Here, we report significant transcriptomic signatures of Cas13bt3 expression in retinal cells and show all-in-one AAV gene therapy with Cas13bt3 can effectively silence VEGFA mRNA in human retinal organoids and humanized VEGF transgenic mouse (trVEGF029, Kimba) models. Specifically, human embryonic stem cells (hESC)-derived retinal pigment epithelium cells show high expression of Cas13bt3 from virus delivery corresponding to a significant reduction of VEGFA mRNA. We further show that intravitreal delivery of Cas13bt3 by AAV2.7m8 can efficiently transduce mouse retinal cells for specific knockdown of human VEGFA in the Kimba mouse. Our results reveal important considerations for assessing Cas13 activity, and establish the Cas13bt3 RNA editing system as a potential anti-VEGF agent that can achieve significant control of VEGFA for the treatment of retinal neovascularization.

AI snapshot

Written from the paper’s full text.

TL;DR

Cas13bt3, a compact RNA-targeting CRISPR enzyme, was characterized for VEGFA knockdown in human cells, hESC-derived retinal organoids, and humanized Kimba mice. AAV2.7m8-delivered Cas13bt3 achieved guide-dependent VEGFA silencing and reduced neovascular signs, but nontargeting sgRNA controls caused substantial transcriptomic changes, especially in retinal cells.

Key findings

  • In HEK293FT cells, shRNA achieved up to 70% VEGFA mRNA knockdown, while CasRx and Cas13bt3 achieved up to 90% knockdown; only Cas13bt3 significantly reduced VEGFA protein in supernatant (P < 0.05).
  • Cas13bt3 with a nontargeting sgRNA produced more DEGs than Cas13bt3 alone or Cas13bt3-VEGFA sgRNA (81 vs 27 and 23, respectively), indicating that NT sgRNA is not an appropriate specificity control.
  • In retinal organoids, AAV2.7m8 transduction efficiency was 18.78% for Cas13bt3-VEGFA sgRNA (1,403/7,467 cells) and 25.95% for Cas13bt3-NT sgRNA (2,260/8,706 cells); RPE cells showed >50% Cas13bt3 positivity in the VEGFA sgRNA group (153/264 cells), while bipolar cells showed ≤10%.
  • In Kimba mice, AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 reduced human VEGFA mRNA by ~50% (P < 0.001) without reducing mouse Vegfa, and treated eyes showed significantly lower leaky vessel density and branch points versus baseline (P < 0.05).

Methods

  • In vitro: HEK293FT cells were transfected with all-in-one AAV plasmids carrying shRNA, CasRx, or Cas13bt3; VEGFA mRNA/protein were measured by RT-qPCR/ELISA, and bulk RNA-seq assessed DEGs, collateral activity, and predicted off-targets.
  • Ex vivo: hESC-derived retinal organoids were transduced with AAV2.7m8.U6.NTsgRNA.Cas13bt3 or AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 and analyzed by scRNA-seq ~6 wk after treatment.
  • In vivo: 6-week-old Kimba mice received bilateral intravitreal AAV2.7m8 vectors; 8 wk later, retinas were analyzed by RT-qPCR, RNA-seq, immunohistochemistry, fundus fluorescein angiography, and OCT. Statistical tests included one-way ANOVA with Tukey’s test, Welch’s t test, Kruskal–Wallis, and Spearman correlation; DEGs used P < 0.05 and |log2 fold change| ≥ 1.

Limitations

  • Stated: the authors did not extensively investigate the stress responses observed in retinal cells, could not extensively study phenotype recovery because of severe disease in the mouse model, and could not confirm VEGFA protein reduction in mouse retinas due to insufficient protein.
  • Stated: overall organoid transduction was determined using an arbitrary cutoff of Cas13bt3 read counts ≥ 10.
  • Evident from the text: significant ex vivo VEGFA knockdown was confirmed only in RPE cells, and the study notes that lower Cas13bt3 copies in other retinal cells may explain the lack of significant knockdown there.

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

Integrity signals

Registry

Crossref · 10.1073/pnas.2408345121

Cites

60 of 60 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

32 of 42 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Initial in vitro studies show promising efficiency and safety of Cas13bt3.

    “however initial in vitro studies show promising efficiency and safety (13, 16).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [13] H. Tong, High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat. Biotechnol. 41, 108– 119 (2022). 10.1038/s41587-022-01419-7. 35953673 source
    • cites [16] X. Deng, Structural basis for the activation of a compact CRISPR-Cas13 nuclease. Nat. Commun. 14, 5845 (2023). 37730702 PMC10511502 source

      The Results section, “Structural-guided engineering of Cas13bt3” (page unavailable), reports that the Y647A variant retained substantial on-target cleavage while greatly reducing collateral cleavage in biochemical assays; cellular assays also showed robust on-target activity with minimal collateral effects. That supports a promising early efficiency and safety-related finding for the engineered variant, but not for Cas13bt3 generally: the same section reports significant collateral degradation with wild-type Cas13bt3, and it does not establish safety beyond these assays. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    A previously described CasRx RNA editing system for ocular neovascularization was validated in vitro in HEK293FT and MIO-M1 cells, demonstrating more than 90% and 50% VEGFA mRNA knockdown, respectively.

    “This approach was validated in vitro in HEK293FT and MIO-M1 cells, demonstrating more than 90% and 50% VEGFA mRNA knockdown, respectively (19).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Y.-F. Chuang, Methods for in vitro CRISPR/CasRx-mediated RNA editing. Front. Cell Dev. Biol. 9, 667879 (2021). 34178991 PMC8226256 source

      The Results section (Figure 3; page unavailable) confirms in vitro VEGFA mRNA testing in HEK293FT and MIO-M1 cells. It reports 47 ± 4% and 68 ± 3% knockdown for the all-in-one constructs in HEK293FT cells, not more than 90%; it describes suppression in MIO-M1 cells without giving the claimed 50% figure. A separate HEK293FT experiment reports 90 ± 2% with a single pre-sgRNA, but that does not establish the stated pair of percentages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.
  • Findingown data

    The highest number of DEGs was from the Cas13bt3-NT sgRNA group compared to Cas13bt3 only or Cas13bt3-VEGFA sgRNA (81 vs 27 and 23).

    “Interestingly, we noted the highest number of differentially expressed genes (DEGs) from the Cas13bt3-NT sgRNA group compared to Cas13bt3 only or Cas13bt3-VEGFA sgRNA (81 vs 27 and 23, respectively; Fig. 1I), a finding that corresponds with recent studies (16, 22).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [16] X. Deng, Structural basis for the activation of a compact CRISPR-Cas13 nuclease. Nat. Commun. 14, 5845 (2023). 37730702 PMC10511502 source

      The Results section (‘Structural-guided engineering of Cas13bt3’) reports collateral RNA degradation in mammalian cells, which supports a general connection to unintended Cas13bt3 effects. It does not report differentially expressed gene counts or compare a non-targeting sgRNA group with Cas13bt3 alone or a VEGFA-targeting group. The 81, 27, and 23 counts are the citing authors’ finding, not a result established by this source. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [22] Z. Li, Intrinsic targeting of host RNA by Cas13 constrains its utility. Nat. Biomed. Eng. 8, 177– 192 (2023). 10.1038/s41551-023-01109-y. 37872368 source
  • Premisecitation

    Significant unintended editing has been reported for CasRx, hampering its clinical progress.

    “however, significant unintended editing has been reported hampering its clinical progress (8, 9)”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [8] Y. Li, Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice. bioRxiv [Preprint] (2022). 10.1101/2022.01.17.476700 (Accessed 23 January 2022). source

      The Abstract reports unintended collateral cleavage of 28S rRNA by RfxCas13d, mouse deaths, and a need for safety evaluation before clinical treatment. The Discussion likewise warns about treatment safety. The paper describes RNA cleavage, not unintended editing, and does not establish that clinical progress has actually been hampered. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [9] P. Shi RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells. bioRxiv [Preprint] (2021). 10.1101/2021.11.30.470032 (Accessed 11 January 2022). source

      The Results report substantial unintended, transcriptome-wide RNA degradation by RfxCas13d in human cells and call for caution in its use for targeted RNA knockdown. They describe collateral RNA degradation rather than unintended editing, and do not establish that it has hampered clinical progress; the paper instead discusses potential therapeutic development. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
  • Study design and conductcitation

    hESC-derived retinal organoids contain all the major cell types in the retina.

    “Here, we used our well-established 2D/3D differentiation protocol to generate hESC-derived retinal organoids containing all the major cell types in the retina (SI Appendix, Fig. S6) (26).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [26] E. L. West, Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids. Stem Cell Rep. 17, 775– 788 (2022). PMC9023802 35334217 source

      The Results section ('Efficient generation of human PSC-derived photoreceptors bearing outer segment-like structures'; page unavailable) describes generating hESC-derived retinal organoids and documents their photoreceptors. The Discussion also identifies rod and cone photoreceptors. Our copy does not establish that these organoids contain all the major retinal cell types. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Classified as probably not a substantial claim. 13
  • May combine two claims. 42

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

    Premisecitation

    The exact specificity profile of CasRx remains contested.

    “and the exact specificity profile of CasRx remains contested (10).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [10] J. F. Bot, J. van der Oost, N. Geijsen, The double life of CRISPR–Cas13. Curr. Opin. Biotechnol. 78, 102789 (2022). 36115160 source

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

    Premisecitation

    RNA editing with CRISPR-Cas13 has emerged as a powerful tool for transient control of gene expression, although recurrent reports of collateral activity are a hurdle for clinical translation.

    “RNA editing with CRISPR-Cas13 has emerged as a powerful tool for transient control of gene expression, although recurrent reports of collateral activity are a hurdle for clinical translation.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [8] Y. Li, Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice. bioRxiv [Preprint] (2022). 10.1101/2022.01.17.476700 (Accessed 23 January 2022). source
    • cites [9] P. Shi RNA-guided cell targeting with CRISPR/RfxCas13d collateral activity in human cells. bioRxiv [Preprint] (2021). 10.1101/2021.11.30.470032 (Accessed 11 January 2022). source

      The Results report collateral RNA degradation by RfxCas13d in human cells and call for caution in using it for targeted RNA knockdown, supporting the concern about collateral activity. They do not establish the claim that RNA editing provides transient control of gene expression or explicitly assess clinical translation. The paper also describes earlier eukaryotic studies as finding no collateral activity and later reports as conflicting. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Repeated intravitreal injections are invasive and risk potentially blinding complications.

    “However, repeated intravitreal injections are invasive and risk potentially blinding complications (18).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] M. Xu, R. Fan, X. Fan, Y. Shao, X. Li, Progress and challenges of anti-VEGF Agents and their sustained-release strategies for retinal Angiogenesis. Drug Des. Devel Ther. 16, 3241– 3262 (2022). PMC9512290 36172053 source
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • Classified as probably not a substantial claim.

    Premisecitation

    Cas13bt3's small size enables applications such as RNA base editing using a single AAV.

    “The newfound enzyme has been reported to possess high specificity and its small size enables further applications such as RNA base editing using a single AAV (13–15).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [13] H. Tong, High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat. Biotechnol. 41, 108– 119 (2022). 10.1038/s41587-022-01419-7. 35953673 source
    • cites [14] G. Li, Mini-dCas13X–mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy. J. Clin. Invest. 133, e162809 (2023). 36512423 PMC9888377 source

      The Results section says the hypercompact mxABE RNA base editor can be packaged in an AAV vector for in vivo delivery. But this paper studies a mini-dCas13X/Cas13X.1-based editor, not Cas13bt3, so it does not establish the claim about Cas13bt3’s size. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [15] Q. Xiao, Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Sci. Transl. Med. 14, eabn0449 (2022). 35857824 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Cas13bt3 has been reported to possess high specificity.

    “The newfound enzyme has been reported to possess high specificity and its small size enables further applications such as RNA base editing using a single AAV (13–15).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [13] H. Tong, High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat. Biotechnol. 41, 108– 119 (2022). 10.1038/s41587-022-01419-7. 35953673 source
    • cites [14] G. Li, Mini-dCas13X–mediated RNA editing restores dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy. J. Clin. Invest. 133, e162809 (2023). 36512423 PMC9888377 source

      The Results sections report high editing specificity for mxABE, a mini-dCas13X–based editor, in cells and mice. Our copy does not address the specificity of Cas13bt3; a search for “Cas13bt3” found no passage about that enzyme. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [15] Q. Xiao, Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Sci. Transl. Med. 14, eabn0449 (2022). 35857824 source

  • Classified as probably not a substantial claim.

    Premisecitation

    Intravitreal anti-VEGF drugs are the approved front-line treatment for DR and nAMD.

    “Intravitreal administration of anti-VEGF drugs is the approved front-line treatment modality for these patients (17).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [17] F.-L. Lin, Gene therapy intervention in neovascular eye disease: A recent update. Mol. Ther. 28, 2120– 2138 (2020). 32649860 PMC7544979 source
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • Classified as probably not a substantial claim.

    Premisecitation

    Hypercompact Cas13bt3 (Cas13X) enzymes range at 775 amino acids.

    “More recently, hypercompact Cas13bt3 (also reported as Cas13X) enzymes were described, ranging even smaller at 775 amino acids (11, 12).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [11] C. Xu, Programmable RNA editing with compact CRISPR–Cas13 systems from uncultivated microbes. Nat. Methods 18, 499– 506 (2021). 33941935 source

      The Main Text (page unavailable) reports exceptionally small Cas13 proteins ranging from 775 to 803 amino acids. It identifies them as Cas13e and Cas13f, however, and does not establish that Cas13bt3 (Cas13X) is 775 amino acids long. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [12] S. Kannan, Compact RNA editors with small Cas13 proteins. Nat. Biotechnol. 40, 194– 197 (2021). 10.1038/s41587-021-01030-2. 34462587 PMC8929162 source

      The Abstract describes Cas13bt proteins as ultra-compact and gives 775–804 amino acids for the smallest members; it also identifies Cas13bt3 as one of the proteins studied. It does not establish that Cas13bt3 specifically is 775 amino acids or that Cas13bt3 is also called Cas13X. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The trVEGF029 (Kimba) mouse is a humanized transgenic model where the neuroretina overexpresses hVEGFA, leading to neovascularization like human proliferative DR.

    “To this end, we used the trVEGF029 (VEGFA+/-, Kimba) mouse, a humanized transgenic mouse model where the neuroretina overexpresses hVEGFA, leading to neovascularization like human proliferative DR (33).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] E. P. Rakoczy, Characterization of a mouse model of hyperglycemia and retinal neovascularization. Am. J. Pathol. 177, 2659– 2670 (2010). 20829433 PMC2966820 source

      The Discussion identifies a human VEGF transgene in Kimba photoreceptors, and the paper reports that transient hVEGF up-regulation causes retinal neovascularization. However, the Discussion describes Kimba’s changes as similar to human nonproliferative DR by eight weeks and says preretinal neovascularization was not observed. The comparison to human proliferative DR therefore overstates the model’s resemblance. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • Rests on an assumption: Overexpression of VEGFA leads to neovascularization and vision loss.

  • May combine two claims.

    Interpretationcitation

    AAV2.7m8 is effective as a vector for retinal transduction and supports its clinical utility.

    “confirming its effectiveness as a vector for retinal transduction and supporting its clinical utility (28, 31).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [28] M. Garita-Hernandez, AAV-mediated gene delivery to 3D retinal organoids derived from human induced pluripotent stem cells. Int. J. Mol. Sci. 21, 994 (2020). 32028585 PMC7036814 source

      The Results (§2.2; page unavailable) report that AAV2-7m8 had the highest transduction efficiency in human retinal organoids. The Conclusions (§5; page unavailable) describe the organoids as an in-vitro model for predicting gene-therapy efficacy, but do not establish AAV2-7m8’s clinical utility in patients. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [31] M. E. McClements, Tropism of AAV vectors in photoreceptor-like cells of human iPSC-derived retinal organoids. Trans. Vision Sci. Technol. 11, 3 (2022). PMC8994202 35377942 source

      The Discussion reports robust AAV2 7m8 transduction in retinal organoids with the CAG promoter, and notes that the vector is being tested for safety and efficacy in humans. These preclinical results support its potential clinical use, but the paper does not establish clinical utility or effectiveness in patients; it also reports minimal expression with the EFS promoter. The indexed passages provide no page numbers. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    The differences in transduction among retinal cell types are likely due to cell-specific transduction, as RPE cells are readily transduced by a variety of AAV capsids, particularly AAV2.7m8.

    “These differences are likely due to cell-specific transduction as RPE cells are readily transduced by a variety of AAV capsids, particularly AAV2.7m8 (29).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [29] T. T. Duong, Comparative AAv-eGFP transgene expression using vector serotypes 1–9, 7m8, and 8b in human pluripotent stem cells, rpes, and human and rat cortical neurons. Stem Cells Int. 2019, 7281912 (2019). 30800164 PMC6360060 source

  • May combine two claims.

    Premisecitation

    The efficacy of gene editing treatments for anti-VEGF is limited by delivery vehicles, poor efficiency of compact Cas9 nucleases, and risk of genomic alterations from long-term Cas9 expression.

    “However, the efficacy of such treatments is limited by the delivery vehicles, the poor efficiency of compact Cas9 nucleases, and the risk of genomic alterations from the long-term expression of Cas9 (46).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [46] S. H. Chung, CRISPR-based VEGF suppression using paired guide RNAs for treatment of choroidal neovascularization. Mol. Ther. Nucleic Acids 28, 613– 622 (2022). 35614998 PMC9114159 source

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

    Study design and conductcitation

    Overall transduction was determined using an arbitrary cut-off (Cas13bt3 read counts ≥ 10) based on previous experience with retinal organoids.

    “It should also be noted that overall transduction was determined using an arbitrary cut-off (Cas13bt3 read counts ≥ 10) based on our previous experience with the retinal organoids (26).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] E. L. West, Antioxidant and lipid supplementation improve the development of photoreceptor outer segments in pluripotent stem cell-derived retinal organoids. Stem Cell Rep. 17, 775– 788 (2022). PMC9023802 35334217 source

      The Results section on AAV-mediated gene supplementation reports transduction of retinal organoids and estimates its efficiency using GFP-positive cells. It does not describe Cas13bt3 read counts or a threshold of ≥10, so it supports the cited prior experience with organoid transduction but not the stated cut-off. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Kimba mice show chronic disease and all major signs of proliferative retinopathy.

    “it shows chronic disease and all major signs of proliferative retinopathy (33).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] E. P. Rakoczy, Characterization of a mouse model of hyperglycemia and retinal neovascularization. Am. J. Pathol. 177, 2659– 2670 (2010). 20829433 PMC2966820 source

      The Discussion says Kimba mice have retinal changes by 8 weeks that remain relatively stable over time, supporting persistent disease. The Clinical and Morphometric Analyses report neovascularization and other retinopathic signs, but say hemorrhages occurred only in Akimba mice and retinal edema affected only a minority of Kimba mice. Thus the claim that Kimba mice show all major signs is too broad. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    RNA editing is generally considered safer than DNA-targeted options as unwanted editing is not permanently retained in the host.

    “While RNA editing is generally considered safer than DNA-targeted options as unwanted editing is not permanently retained in the host (47)”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [47] B. J. Booth, RNA editing: Expanding the potential of RNA therapeutics. Mol. Ther. 31, 1533– 1549 (2023). 36620962 PMC9824937 source

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

    Premisecitation

    Recent efforts to address the problem of frequent intravitreal injections have employed gene editing strategies like targeting VEGFA using CRISPR-Cas9.

    “Recent efforts to address this problem have employed gene editing strategies like targeting VEGFA using CRISPR-Cas9 (43, 45).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [43] J. Park, CRISPR/Cas9 mediated specific ablation of vegfa in retinal pigment epithelium efficiently regresses choroidal neovascularization. Sci. Rep. 13, 3715 (2023). 36878916 PMC9988861 source
    • cites [45] X. Huang, Genome editing abrogates angiogenesis in vivo. Nat. Commun. 8, 112 (2017). 28740073 PMC5524639 source

      The Introduction and Discussion describe AAV-mediated CRISPR-Cas9 editing as a potential alternative to anti-VEGF treatment that requires chronic administration. But this study edits genomic VEGFR2, not VEGFA; the claimed target is incorrect. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The major concern with current anti-VEGF treatment is the need for frequent intravitreal injections.

  • May combine two claims.

    Findingown data

    Cas13bt3 results show significant knockdown of VEGFA mRNA in human cell lines without impact on predicted off-target genes.

    “Promisingly, our results show a significant knockdown of VEGFA mRNA in human cell lines without impact on predicted off-target genes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Findingown data

    In vivo gene therapy showed robust VEGFA mRNA silencing in mouse models of proliferative retinopathy along with control of neovascularization symptoms.

    “Last, in vivo gene therapy showed robust VEGFA mRNA silencing in mouse models of proliferative retinopathy along with control of neovascularization symptoms.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.
    • Rests on an assumption: Overexpression of VEGFA leads to neovascularization and vision loss.

  • May combine two claims.

    Findingown data

    sgRNA2 yielded little to no VEGFA knockdown while sgRNA3 exhibited ~30% knockdown.

    “Compared to the original (referred to here as sgRNA1), both sgRNA2 and sgRNA3 had poorer efficiency, with sgRNA2 yielding little to no VEGFA knockdown while sgRNA3 exhibited ~30% knockdown.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Findingown data

    None of the top 10 candidate off-target genes were significantly downregulated, indicating a lack of off-target activity from Cas13bt3.

    “Promisingly, none of the off-target genes were significantly downregulated, indicating a lack of off-target activity from Cas13bt3, as was the case for shRNA and CasRx (SI Appendix, Fig. S2E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Significant VEGFA mRNA knockdown was observed from all three RNA-targeting methods, with up to 70% shRNA and up to 90% CasRx and Cas13bt3 compared to vector controls.

    “Significant VEGFA mRNA knockdown was observed from all three RNA-targeting methods, with up to 70% shRNA VEGFA mRNA knockdown (P < 0.001), while CasRx and Cas13bt3 achieved up to 90% knockdown (P < 0.0001) compared to vector controls (AAV plasmid carrying a fluorescent marker) (Fig. 1F; n = 4 independent experiments).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Findingown data

    Cas13bt3 demonstrates potent VEGFA knockdown in vitro in human cell lines, 3D hESC-derived retinal organoids, and in vivo in humanized VEGF transgenic mouse retinae.

    “Here, we used Cas13bt3 to demonstrate potent VEGFA knockdown in vitro in human cell lines, 3D human embryonic stem cells (hESC)-derived retinal organoids, and in vivo in humanized VEGF transgenic mouse retinae.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Findingown data

    Bipolar cells showed the least expression of Cas13bt3 (≤10%), while all other cell types showed moderate expression (≤25%).

    “Bipolar cells showed the least expression of Cas13bt3 (≤ 10%), while all other cell types showed moderate expression (≤25%) (Fig. 2C and SI Appendix, Fig. S10).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Macroglial and bipolar cells exhibited the highest DEGs, especially higher in the Cas13bt3-NT sgRNA group.

    “We found macroglial (indicating Müller cells in this population) and bipolar cells exhibited the highest DEGs, which were especially higher in the Cas13bt3-NT sgRNA group (Macroglial–NT sgRNA: 5571 vs VEGFA sgRNA: 2928, Bipolar–NT sgRNA: 3725 vs VEGFA sgRNA: 1633) (Fig. 2F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DEG levels were comparable in both single and multiple sgRNA vectors, suggesting the additional sgRNAs do not increase overall collateral activity.

    “Interestingly, DEG levels were comparable in both single and multiple sgRNA vectors, suggesting the additional sgRNAs do not increase overall collateral activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RPE and macroglial cells expressed high levels of Cas13bt3 while most cones, rods, and horizontal and bipolar cells only showed low levels of Cas13bt3 expression.

    “Here, we found that RPE and macroglial cells expressed high levels of Cas13bt3 expression while most cones, rods, and horizontal and bipolar cells only showed low levels of Cas13bt3 expression (Fig. 2H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    RNA sequencing confirmed that mVegfa expression and predicted off-target genes were not significantly altered, indicating low off-target activity from Cas13bt3-mediated RNA silencing.

    “RNA sequencing further confirmed that mVegfa expression and predicted off-target genes were not significantly altered, indicating low off-target activity from Cas13bt3-mediated RNA silencing (SI Appendix, Fig. S15).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    DEGs for both single and sgRNA array groups were comparable, suggesting minimal collateral activity.

    “To account for perturbations from the NT sgRNA, we compared DEGs to the NT sgRNA group and found DEGs for both single and sgRNA array groups were comparable, suggesting minimal collateral activity (SI Appendix, Fig. S5 A and B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HEK293FT appears resistant to high levels of collateral activity, while retinal cells exhibited significantly higher levels of transcriptomic effects.

    “While HEK293FT appears resistant to high levels of collateral activity, retinal cells exhibited significantly higher levels of transcriptomic effects.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV2.7m8.CMV.mCherry-treated retinal flatmounts showed high levels of fluorescence through all retinal layers 4 wk following injections, confirming quick onset and deep penetration of the retina from intravitreal delivery.

    “Immunohistochemistry analysis of AAV2.7m8.CMV.mCherry-treated retinal flatmounts showed high levels of fluorescence through all retinal layers (RGCs, bipolar and photoreceptor cells) 4 wk following injections, confirming quick onset and deep penetration of the retina from intravitreal delivery (SI Appendix, Fig. S14 C and D; n = 6 eyes from 3 animals).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cas13bt3-mediated RNA silencing significantly reduces VEGFA mRNA expression, specifically targeting hVEGFA and controlling hallmark signs of neovascularization in a humanized mouse model.

    “Overall, our findings showed that Cas13bt3-mediated RNA silencing significantly reduces VEGFA mRNA expression, specifically targeting hVEGFA and controlling hallmark signs of neovascularization in a humanized mouse model.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.
    • Rests on an assumption: Overexpression of VEGFA leads to neovascularization and vision loss.

  • May combine two claims.

    Findingown data

    A significant number of DEGs were observed across all cell types in the retinal organoid that received AAV2.7m8.U6.NTsgRNA.Cas13bt3, with most DEGs upregulated.

    “Here, we observed a significant number of DEGs across all cell types in the retinal organoid that received the AAV2.7m8.U6.NTsgRNA.Cas13bt3, where most DEGs were upregulated genes (Fig. 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    No reduction of hVEGFA or mVegfa was observed with AAV2.7m8.U6.NTsgRNA.Cas13bt3 or control groups, indicating guide-dependent silencing of hVEGFA mRNA.

    “No reduction of hVEGFA or mVegfa was observed with AAV2.7m8.U6.NTsgRNA.Cas13bt3 or control groups, indicating guide-dependent silencing of hVEGFA mRNA (Fig. 3 F and G; n = 3 to 12 retinas).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Findingown data

    Among retinal cell types studied, bipolar cells showed the least transduction by AAV2.7m8, while RPE cells seemed more susceptible.

    “Among the retinal cell types studied, bipolar cells showed the least transduction by AAV2.7m8 while RPE cells seemed more susceptible.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    No significant changes in total retinal thickness were observed between untreated and treated groups, and there was a general decline in TRT across all animals.

    “However, we observed no significant changes between the untreated and treated groups and a general decline in TRT across all animals (SI Appendix, Fig. S19).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 treated eyes showed significantly lower leaky vessel density and branch points compared to baseline at 8 wk posttreatment, unlike the control and the AAV2.7m8.U6.NTsgRNA.Cas13bt3 eyes.

    “The AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 treated eyes showed significantly lower leaky vessel density and branch points compared to baseline at 8 wk posttreatment, unlike the control and the AAV2.7m8.U6.NTsgRNA.Cas13bt3 eyes (Fig. 3 J and K and SI Appendix, Fig. S18, P < 0.05; n = 4 to 13 retinas).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.
    • Rests on an assumption: Overexpression of VEGFA leads to neovascularization and vision loss.

  • May combine two claims.

    Findingown data

    The same pattern of DEGs was observed in the VEGFA sgRNA group, albeit at a lower extent, suggesting Cas13bt3-NT sgRNA exhibited a greater impact on the transcriptome.

    “The same pattern was observed in the retinal organoid transduced with AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 (Fig. 2E), albeit at a lower extent, suggesting that the Cas13bt3-NT sgRNA exhibited a greater impact on the transcriptome.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Vessel density and branching were noticeably reduced in the mouse model.

    “while vessel density and branching were noticeably reduced”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Average transduction efficiency was 18.78% for the Cas13bt3-VEGFA sgRNA group and 25.95% for the Cas13bt3-NT sgRNA group.

    “Using this threshold, we determined an average transduction efficiency of 18.78% (1,403 out of 7,467 cells) for the Cas13bt3-VEGFA sgRNA group and 25.95% (2,260 out of 8,706 cells) for the Cas13bt3-NT sgRNA group.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Following RNA editing gene therapy in Kimba retinae, specific knockdown of human VEGFA was demonstrated, without impact on endogenous mouse Vegfa.

    “Following RNA editing gene therapy in Kimba retinae, we demonstrated the specific knockdown of human VEGFA, without impact on endogenous mouse Vegfa.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There was ~50% reduction of human VEGFA mRNA but not mouse Vegfa in the AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 group.

    “This corresponded with ~50% reduction (P < 0.001) of human VEGFA (hVEGFA) mRNA (Fig. 3F) but not mouse Vegfa (mVegfa) (Fig. 3G) in the AAV2.7m8.U6.VEGFAsgRNA.Cas13bt3 group.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: VEGF is a key mediator of ocular neovascularization.
    • Rests on an assumption: Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

  • May combine two claims.

    Interpretationown data

    Overexpression of hVEGFA promotes rapid retinal degeneration that could not be controlled or reversed through VEGFA silencing.

    “We postulate that the overexpression of hVEGFA promotes rapid retinal degeneration that could not be controlled or reversed through VEGFA silencing.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Overexpression of VEGFA leads to neovascularization and vision loss.

  • May combine two claims.

    Interpretationown data

    Intravitreal injection with all-in-one AAV Cas13bt3 can achieve potent silencing of VEGFA, addressing drawbacks with current gene editing strategies.

    “Our results indicate that intravitreal injection with all-in-one AAV Cas13bt3 can achieve potent silencing of VEGFA, addressing drawbacks with current gene editing strategies.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The major concern with current anti-VEGF treatment is the need for frequent intravitreal injections.

  • May combine two claims.

    Interpretationown data

    Laminin-related processes were strongly downregulated across all groups (Cas13bt3 only, Cas13bt3-NT sgRNA, and Cas13bt3-VEGFA sgRNA), suggesting intrinsic targets could exist for Cas13bt3.

    “Here, we found that laminin-related processes (e.g., “laminin binding”, “laminin complex”) were strongly downregulated across all groups (Cas13bt3 only, Cas13bt3-NT sgRNA, and Cas13bt3-VEGFA sgRNA) suggesting intrinsic targets could exist for Cas13bt3 as well (Dataset S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The high levels of DEGs observed in various cells from retinal organoids suggest significant inflammatory responses as well as cell-specific collateral activity.

    “This is supported by the high levels of DEGs we observed in various cells from the retinal organoids suggesting significant inflammatory responses as well as cell-specific collateral activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The significant VEGFA knockdown in RPE cells is likely due to preferential transduction of AAV2.7m8 and strong expression of Cas13bt3 in these cells.

    “likely due to preferential transduction of AAV2.7m8 and the strong expression of Cas13bt3 in these cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    With a NT sgRNA, Cas13bt3 initiates an unspecific target-search process involving significant RNA binding that impacts the expression levels of several genes, while a targeting sgRNA narrows the target-search process to reduce overall transcriptomic impact.

    “Our results suggest that with a NT sgRNA, Cas13bt3 initiates an unspecific target-search process involving significant RNA binding that impacts the expression levels of several genes, while a targeting sgRNA narrows the target-search process to reduce overall transcriptomic impact.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The VEGFA sgRNA array could still be processed by Cas13bt3 and competition for crRNA loading did not affect RNA silencing.

    “This showed that the VEGFA sgRNA array could still be processed by Cas13bt3 and competition for crRNA loading did not affect RNA silencing.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Ethics approval was obtained from The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee (22-036-UM).

    “Ethics approval was obtained from The Florey Institute of Neuroscience and Mental Health Animal Ethics Committee (22-036-UM).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The study did not extensively investigate the stress responses observed from retinal cells.

    “we did not extensively investigate the stress responses we observed from the retinal cells.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Animal procedures were approved by the Animal Ethics Committees of University of Melbourne and conducted in accordance with ARVO Statement.

    “All animal procedures were approved by the Animal Ethics Committees of University of Melbourne and conducted in accordance with The Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animal in Ophthalmic and Vision Research.”
    Quote in the paperNot a substantial claim

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

    Study design and conductreasoning

    To account for multiple testing, P-values underwent adjustment through the Benjamini-Hochberg false discovery rate (BH-FDR).

    “To account for multiple testing, P-values underwent adjustment through the Benjamini–Hochberg false discovery rate (BH-FDR).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The study demonstrated that hypercompact Cas13bt3 can be efficiently delivered and expressed in various retinal cell types for effectively controlling VEGFA expression and neovascularization.

    “This study demonstrated that the hypercompact Cas13bt3 can be efficiently delivered and expressed in various retinal cell types for effectively controlling VEGFA expression and neovascularization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The study offers insights into the Cas13 targeting mechanism and its utility in targeting retinal and RPE cells, providing evidence for the potential of Cas13-based therapies for ocular disease.

    “In this study, we offer insights into the Cas13 targeting mechanism and its utility in targeting retinal and RPE cells, providing evidence for the potential of Cas13-based therapies for ocular disease.”
    Quote in the paperSubstantial claim
53 of 53

  • The paper relies on this without support.

    VEGF is a key mediator of ocular neovascularization.

    “Vascular endothelial growth factor (VEGF) has been described as a key mediator of this process, and the overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy (DR) and neovascular age-related macular degeneration (nAMD).”

  • The paper relies on this without support.

    Overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy and neovascular age-related macular degeneration.

    “Vascular endothelial growth factor (VEGF) has been described as a key mediator of this process, and the overexpression of VEGFA underlies pathological angiogenesis in common ocular diseases like diabetic retinopathy (DR) and neovascular age-related macular degeneration (nAMD).”

  • The paper relies on this without support.

    Overexpression of VEGFA leads to neovascularization and vision loss.

    “In disease, overexpression of VEGFA leads to neovascularization and vision loss.”

  • The paper relies on this without support.

    VEGFA is essential for both physiological and pathological angiogenesis.

    “We chose to target the VEGFA gene as this growth factor is essential for both physiological and pathological angiogenesis.”

  • The paper relies on this without support.

    Disease progression in each retina is variable.

    “As disease progression in each retina is variable, we performed paired analyses of recordings from each retina at the endpoint to its baseline.”

  • The paper relies on this without support.

    Retinal organoid models mimic the human retina containing lamination and major retinal cell types of the developing retina.

    “Retinal organoid models enable the testing of therapies in an environment that mimics the human retina containing lamination and major retinal cell types of the developing retina.”

  • The paper relies on this without support.

    VEGFA silencing in one cell type may have collateral effects on other retinal cells.

    “as VEGFA silencing in one cell type may have collateral effects on other retinal cells.”

  • The paper relies on this without support.

    Preclinical studies with Cas13 nucleases are commonly performed in HEK293FT cells, and its specificity has often been exaggerated in the literature.

    “As preclinical studies with Cas13 nucleases are commonly performed in HEK293FT cells, its specificity has often been exaggerated in the literature.”

  • The paper relies on this without support.

    RPE cells are the main producers of VEGFA in nAMD.

    “with RPE cells being the main producers of VEGFA in nAMD”

  • The paper relies on this without support.

    Kimba mice express human VEGFA.

    “as Kimba mice express human VEGFA”

  • The paper relies on this without support.

    The major concern with current anti-VEGF treatment is the need for frequent intravitreal injections.

    “The major concern with current anti-VEGF treatment is the need for frequent intravitreal injections.”

  • The paper relies on this without support.

    RNA-targeting molecules like Cas13 require persistent expression for therapeutic effect.

    “As RNA-targeting molecules like Cas13 require persistent expression for therapeutic effect”
12 of 12

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Overall NT-sgRNA transduction exceeds every reported cell-type rate

    Minor

    The reported overall transduction rate for the Cas13bt3-NT sgRNA group is 25.95%. Yet the paper says its RPE rate is about 15%, its bipolar rate is at most 10%, and all other cell-type rates are at most 25%; their combined rate cannot exceed 25%.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. S. Andrews, FastQC: A Quality Control Tool for High Throughput Sequence Data (Babraham Bioinformatics, Babraham Institute, Cambridge, United Kingdom, 2010).
  2. G.-S. Liu Cas13bt3 knockdown of VEGFA mRNA in retinal organoid [scRNA-Seq]. Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE259219. Deposited 1 March 2024.
  3. G.-S. Liu Cas13bt3 knockdown of VEGFA mRNA in mammalian cells and mouse retina [RNA-Seq]. Gene Expression Omnibus. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE259220. Deposited 1 March 2024.

Cited by

Sorted by citations

Related

Authors
Kumar, S., Hsiao, Y.-W., Wong, V. H. Y., Aubin, D., Wang, J.-H., Lisowski, L., Rakoczy, E. P., Li, F., Alarcon-Martinez, L., Gonzalez-Cordero, A., Bui, B. V., & Liu, G.-S.
Affiliations
  • University of Tasmania (AU)
  • The Royal Victorian Eye & Ear Hospital (AU)
  • The University of Melbourne (AU)
  • The University of Sydney (AU)
  • The University of Western Australia (AU)
  • Zhongshan People's Hospital (CN)
  • Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC 3002, Australia
Corresponding author
Liu, Guei-Sheung
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2024-10-30
Volume
121
Issue
45
Pages
e2408345121-e2408345121
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2408345121
PMC
PMC11551378
OpenAlex
W4403917595
Cited by
19
References
59
Citations by year
2026: 10 · 2025: 7 · 2024: 2
FWCI
2.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • Minerva Access (University of Melbourne) (submittedVersion, cc-by-nc-nd)
Funders
  • DHAC | National Health and Medical Research Council (1185600, MRF2008912)
  • MOST | National Natural Science Foundation of China (82201227)
  • National Natural Science Foundation of China-Guangdong Joint Fund (2023A1515011225)
  • National Stem Cell Foundation of Australia (00339)
  • Rebecca L. Cooper Medical Research Foundation (n/a)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
CRISPR-Cas13, RNA editing, adeno-associated virus gene therapy, anti-VEGF therapy, retinal neovascularization, retinal pigment epithelium, intravitreal delivery, retinal organoids, gene silencing
MeSH terms
Dependovirus, Animals, Humans, Mice, Transgenic, Retina, Genetic Therapy, RNA Editing, Vascular Endothelial Growth Factor A, Mice, Retinal Pigment Epithelium, CRISPR-Cas Systems

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 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

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