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Cited by 876CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Open access
Yin, Hao, Song, Chun-Qing, Dorkin, Joseph R, Zhu, Lihua J, Li, Yingxiang, Wu, Qiongqiong, Park, Angela, Yang, Junghoon +14 more·2016·Nature Biotechnology
DOI 10.1038/nbt.3471PMC PMC5423356
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Abstract

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

AI snapshot

Written from the paper’s full text.

TL;DR

This study combined lipid nanoparticle delivery of Cas9 mRNA with AAV-delivered sgRNA and an HDR template to correct the Fah mutation in a mouse model of hereditary tyrosinemia type I. A single treatment corrected >6% of hepatocytes, restored Fah splicing, and rescued weight loss and liver damage.

Key findings

  • In Fahmut/mut mice, combined nano.Cas9 + AAV-HDR treatment yielded 6.2% ± 1.0% Fah-positive hepatocytes by immunohistochemistry after a single application (n = 4 mice), with correction correlating with nano.Cas9 and AAV-HDR dose.
  • Treated mice had 9.5% restored FAH mRNA without selection, 24.1% average indels at the sgRNA target region, and 0.81% ± 0.08% corrected “G-CC” sequence in total liver DNA by deep sequencing.
  • Treatment completely prevented body weight loss after NTBC withdrawal, all nano.Cas9 + AAV-HDR mice survived 30 days, and serum AST, ALT, and bilirubin indicated substantially reduced liver damage compared with controls.
  • Off-target editing was low at assayed sites: <0.3% indels at three top predicted off-target sites, and in vivo OT1/GOT sites were not substantially higher than background (P > 0.05); transient mRNA Cas9 also reduced off-target editing versus long-term lentiviral Cas9 in cells.

Methods

  • Fahmut/mut mice (8–10 weeks old, male and female) were injected via tail vein with AAV2/8-HDR (6 × 10^11 or 1.2 × 10^12 genome copies) and nano.Cas9 (1 or 2 mg/kg); in the efficacy experiment, AAV-HDR was given at day −14, nano.Cas9 at day −7, and NTBC water was withdrawn at day 0.
  • Cas9 mRNA was chemically modified and formulated with C12-200, cholesterol, C14PEG2000, DOPE, and arachidonic acid in lipid nanoparticles; AAV-HDR encoded U6-sgRNA and a 1.7-kb HDR template designed to correct the mutant “A” to wild-type “G” and introduce a “CC” PAM motif.
  • Analyses included liver immunohistochemistry, serum biomarkers, qRT-PCR, Sanger sequencing, Illumina deep sequencing, GUIDE-Seq in Hepa1-6 cells, and statistics by Student’s t-tests and one-way ANOVA with Tukey post-test.

Limitations

  • Stated: non-viral delivery of DNA into the nucleus in vivo with high efficiency and low toxicity remains difficult, and further improvement of HDR efficacy may require optimizing HDR template design and inhibiting the NHEJ pathway.
  • Stated: the stringent “G-CC” pattern counting may underestimate HDR events, and an alternative explanation for the observed data is partial gene conversion; off-target analysis was limited to predicted and assayed sites.
  • Evident from the text: efficacy was shown only in the Fahmut/mut mouse model, with small group sizes (n = 3–4 mice) and 30-day follow-up, so long-term durability, AAV immunogenicity, and human translation are not established.

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

Integrity signals

Registry

Crossref · 10.1038/nbt.3471

Cites

47 of 47 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 40 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Deep sequencing confirmed a corrected 'G-CC' pattern at the Fah locus in 0.81% ± 0.08% of the total liver DNA, in contrast to the AAV-HDR alone group, which contained no detectable 'G-CC' sequences.

    “The analysis of deep sequencing data confirmed a corrected “G-CC” pattern at the Fah locus in 0.81% ± 0.08% of the total liver DNA, in contrast to the AAV-HDR alone group, which contained no detectable “G-CC” sequences (Fig. 3g).”
    Quote in the paperSubstantial claim
  • Findingown data

    Systemic delivery of Cas9 mRNA by lipid nanoparticles and sgRNA/HDR template by AAV can efficiently cure Fahmut/mut mice.

    “Here we report that systemic delivery of Cas9 mRNA by lipid nanoparticles and sgRNA/HDR template by AAV can efficiently cure Fahmut/mut mice.”
    Quote in the paperSubstantial claim
  • Findingown data

    Deep sequencing of the GFP provirus region from genomic DNA (n = 4) observed insertional or deletional mutations surrounding the Cas9 cleavage site.

    “We observed insertional or deletional mutations (indels) surrounding the Cas9 cleavage site (Fig. 1c–e and Supplementary Table 3).”
    Quote in the paperSubstantial claim
  • Findingown data

    The number of Fah-positive hepatocytes correlated with the dose of nano.Cas9 and AAV-HDR.

    “The number of Fah-positive hepatocytes correlated with the dose of nano.Cas9 and AAV-HDR (Fig. 3c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Cas9 protein in total liver lysates was detected by immunoblot after intravenous injection of nano.Cas9 (1 mg/kg or 2 mg/kg).

    “To determine whether lipid nanoparticles can deliver Cas9 mRNA, nano.Cas9 (1 mg/kg or 2 mg/kg) was injected intravenously, and Cas9 protein in total liver lysates was detected by immunoblot (Supplementary Fig. 2d).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 15
  • May combine two claims. 28
  • The quote only partly states this claim. 1

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

    Findingown data

    The mean particle diameter of nano.Cas9 is about 120 nm as determined by dynamic light scattering.

    “The mean particle diameter of nano.Cas9 is about 120 nm as determined by dynamic light scattering (Supplementary Fig. 1c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Compared to indels at the on-target Fah site, <0.3% indels were detected at the assayed off-target sites in nano.Cas9 + AAV-HDR-treated mice, and these numbers were comparable with AAV-HDR-treated control mice.

    “Compared to indels at the on-target Fah site (Fig. 3f), <0.3% indels were detected at the assayed off-target sites in nano.Cas9 + AAV-HDR–treated mice, and these numbers were comparable with AAV-HDR–treated control mice (Supplementary Fig. 7 and Supplementary Table 6), indicating that Cas9 mRNA delivery has minimal off-target effects at assayed sites for Fah sgRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vivo, OT1 and several other assayed sites had modest indel rates, and none of these sites were substantially higher than the background indel rate in untreated livers (P > 0.05).

    “OT1 and several other assayed sites had modest indel rates, and none of these sites were substantially higher than the background indel rate in untreated livers (P > 0.05; Supplementary Fig. 8b and Supplementary Table 9).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Co-injection of an adenovirus encoding Cas9 with AAV-HDR for Ctnnb1 produced ∼24% indels and ∼1.2% beta-Catenin-positive hepatocytes in vivo.

    “We observed ∼24% indels and ∼1.2% beta-Catenin–positive hepatocytes in vivo (Supplementary Fig. 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A PCR approach observed a clear band in livers of nano.Cas9 + AAV-HDR-treated animals, but not in control animals, and sequencing identified that the corrected sequence ('G-CC' pattern) is integrated.

    “We observed a clear band in livers of nano.Cas9 + AAV-HDR–treated animals, but not in control animals (Supplementary Fig. 5b). We sequenced the band and identified that the corrected sequence (“G-CC” pattern) is integrated (Supplementary Fig. 5c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Targeted deep sequencing on nuclease-treated Hepa1-6 cells detected 2.4% and 1.8% indels at OT1 in two replicates, consistent with OT1 being a Cas9:sgFah off-target site in transformed cells in vitro.

    “These additional sites did not meet our peak calling criteria, but we detected 2.4% and 1.8% indels at OT1 in two replicates (Supplementary Fig. 8a and Supplementary Table 9), consistent with OT1 being a Cas9:sgFah off-target site in transformed cells in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cas9 mRNA was present in the liver at 4 h and 14 h but was significantly diminished at 24 h (P < 0.05), consistent with transient expression.

    “The Cas9 mRNA was present in the liver at 4 h and 14 h but was significantly diminished at 24 h (P < 0.05), consistent with transient expression (Supplementary Fig. 2e).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    sgRNA was already expressed at day 3 after AAV-HDR injection, but its levels were more than tenfold higher at day 7 and day 14.

    “We found sgRNA was already expressed at day 3, but its levels were more than tenfold higher at day 7 and day 14.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The efficiency of Fah correction was >6% of hepatocytes after a single application, with up to 6.2% ± 1.0% hepatocytes staining positive for Fah protein in treated animals.

    “The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.”
    • Also stated in: § Abstract · plain
      “Up to 6.2% ± 1.0% hepatocytes stained positive for the Fah protein by immunohistochemistry in nano.Cas9 plus AAV-HDR–treated animals (Fig. 3b,c; n = 4 mice; see Supplementary Table 4 for percentage of Fah-positive cells, age and gender of each mouse).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The particle size of nano.Cas9 was the same on day 0, 7, 11 and 18, indicating these particles are stable for at least 18 d in PBS.

    “The particle size of nano.Cas9 was the same on day 0, 7, 11 and 18 (Supplementary Fig. 1d,e), indicating these particles are stable for at least 18 d in PBS.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    β-gal protein was detected in the mouse liver by immunoblot at 14 h after administration of a single dose (1 mg/kg or 2 mg/kg), and the amount of protein expressed correlated with the dose of mRNA.

    “β-gal protein was detected in the mouse liver by immunoblot at 14 h after administration of a single dose (1 mg/kg or 2 mg/kg), and the amount of protein expressed correlated with the dose of mRNA (Supplementary Fig. 2b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Most indels are frameshift (e.g., 1 nt and 2 nt) mutations, which potentially cause loss of function of the GFP reporter.

    “Most indels are frameshift (e.g., 1 nt and 2 nt) mutations (Fig. 1e), which potentially cause loss of function of the GFP reporter.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Systemic delivery of Cas9 mRNA by lipid nanoparticles, and sgRNA/HDR template by AAV, corrected a Fah mutation and restored Fah splicing in more than 6% of hepatocytes in the adult mouse liver, an order of magnitude improvement over high-pressure injection of DNA.

    “Systemic delivery of Cas9 mRNA by lipid nanoparticles, and sgRNA/HDR template by AAV, corrected a Fah mutation and restored Fah splicing in more than 6% of hepatocytes in the adult mouse liver, an order of magnitude improvement over that previously generated using high-pressure injection of DNA16.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    This treatment is well-tolerated in mice and fully rescued body weight loss and liver damage in tyrosinemia mice.

    “This treatment is well-tolerated in mice and fully rescued body weight loss and liver damage in tyrosinemia mice.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The majority of the cells in liver sections stained positive in a β-gal activity assay, indicating systemic delivery of mRNA to most of the cells in the mouse liver.

    “The majority of the cells in liver sections stained positive in a β-gal activity assay (Supplementary Fig. 2c), indicating systemic delivery of mRNA to most of the cells in the mouse liver.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In HTI patients, mutation of FAH leads to accumulation of toxic metabolites and severe liver damage.

    “In HTI patients, mutation of fumarylacetoacetate hydrolase (FAH), the last enzyme catalyzing the tyrosine catabolic pathway, leads to accumulation of toxic metabolites and severe liver damage8.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    The Fahmut/mut mouse model is caused by a G→A point mutation in the last nucleotide of exon 8, which causes skipping of exon 8 and truncated Fah mRNA.

    “The Fahmut/mut mouse model6,8 is caused by a G→A point mutation in the last nucleotide of exon 8. This causes skipping of exon 8 and truncated Fah mRNA.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [6] Aponte, J.L. et al. Point mutations in the murine fumarylacetoacetate hydrolase gene: animal models for the human genetic disorder hereditary tyrosinemia type 1. Proc. Natl. Acad. Sci. USA 98, 641–645 (2001). source

      Not checked in the free run.

    • cites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationreasoning

    Non-viral delivery of Cas9 mRNA allows for short-term expression and, eventually, complete removal of the nuclease from the body.

    “An alternative approach is the non-viral delivery of Cas9 mRNA, which would allow for short-term expression and, eventually, complete removal of the nuclease from the body.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    All of these delivery studies have reported gene knockout rather than gene repair, a less efficient process requiring a DNA repair template.

    “However, all of these delivery studies have reported gene knockout rather than gene repair, a less efficient process requiring a DNA repair template15.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Chu, V.T., Weber, T. & Wefers, B. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol. 33, 543–548 (2015). source

      Not checked in the free run.

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

    Premisecitation

    Cas9:sgRNA recognizes the PAM sequence and a complementary 20-nucleotide genomic sequence and induces double-strand DNA breaks, which are repaired by error-prone NHEJ or precise HDR.

    “Cas9:sgRNA recognizes the protospacer-adjacent motif (PAM) sequence and a complementary 20-nucleotide genomic sequence and induces double-strand DNA breaks, which are repaired by error-prone nonhomologous end-joining (NHEJ) or precise homology-directed repair (HDR)2,5.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [2] Doudna, J.A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014). source

      Not checked in the free run.

    • cites [5] Sander, J.D. & Joung, J.K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014). source

      Not checked in the free run.

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

    Premisecitation

    A smaller form of Cas9 was developed and shown capable of packing and delivery with sgRNA by AAV in vivo.

    “Recently, a smaller form of Cas9 was developed, and shown capable of packing and delivery with sgRNA by AAV in vivo12.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Lipid and lipid-like formulations have shown great promise as delivery agents for siRNA in a range of species, including humans.

    “Lipid and lipid-like formulations have shown great promise as delivery agents for siRNA in a range of species, including humans19.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Yin, H. et al. Non-viral vectors for gene-based therapy. Nat. Rev. Genet. 15, 541–555 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Hydrodynamic injection yielded a low correction rate of 0.4% of hepatocytes and has been tested in only one human clinical trial.

    “Nevertheless, hydrodynamic injection yielded a low correction rate of 0.4% of hepatocytes, and this method has been tested in only one human clinical trial17.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Yin, H. et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551–553 (2014). source

      Not checked in the free run.

    • cites [17] Khorsandi, S.E. et al. Minimally invasive and selective hydrodynamic gene therapy of liver segments in the pig and human. Cancer Gene Ther. 15, 225–230 (2008). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    AAV has a loading capacity size of approximately 4.7 kb, and the most commonly used S. pyogenes Cas9 is difficult to fit in typical AAV constructs with efficient promoters.

    “However, AAV has a loading capacity size of approximately 4.7 kb, and the most commonly used form of Cas9 from Streptococcus pyogenes is difficult to fit in typical AAV constructs with efficient promoters13.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [13] Swiech, L. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33, 102–106 (2015). source

      Not checked in the free run.

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

    Premisecitation

    Fahmut/mut mice can be treated with NTBC, an inhibitor of an enzyme upstream of Fah, to prevent toxin accumulation in hepatocytes.

    “These mice can be treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), an inhibitor of an enzyme upstream of Fah, to prevent toxin accumulation in hepatocytes8.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [8] Paulk, N.K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51, 1200–1208 (2010). source

      Not checked in the free run.

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

    Premisecitation

    The AAV dose used (up to 1.2 × 10^12 genome copies/mouse) is a standard dose described in the literature.

    “To express sgRNA, we treated animals with up to 1.2 × 1012 AAV genome copies/mouse, a standard dose described in the literature28.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Barzel, A. et al. Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Combining non-viral delivery of Cas9 mRNA and an AAV vector with a sgRNA expression cassette and an HDR template allowed for short-term expression of the Cas9 nuclease, which provided efficient on-target genome editing, while potentially reducing off-target editing.

    “This allowed for short-term expression of the Cas9 nuclease, which provided efficient on-target genome editing, while potentially reducing off-target editing (Fig. 1h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    A smaller form of Cas9 (S. aureus Cas9) fits into AAV vectors and has been used to knockout genes by NHEJ in the mouse liver, with more than 40% indel formations in the Pcsk9 locus reported.

    “A smaller form of Cas9 (Staphylococcus aureus Cas9), which fits into AAV vectors, has been recently used to knockout genes by NHEJ in the mouse liver. More than 40% indel formations in Pcsk9 locus was reported12.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source

      Not checked in the free run.

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

    Study design and conductnone given

    All animal experiments were performed under the guideline of the MIT Animal Care and Use Committee.

    “All animal experiments were performed under the guideline of the MIT Animal Care and Use Committee.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Interpretationreasoning

    The calculation 6% hepatocytes × 0.6/4 = 0.9% estimated DNA is consistent with the 0.81% corrected 'G-CC' sequence pattern observed by deep sequencing.

    “This estimation is consistent with the 0.81% corrected “G-CC” sequence pattern observed by deep sequencing in treated liver samples (Fig. 3g).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Chemical modification of sgRNA improved gene editing in vitro when co-delivered with Cas9 mRNA.

    “A recent study demonstrated that the chemical modification of sgRNA improved gene editing in vitro when co-delivered with Cas9 mRNA32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol. 33, 985–989 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Transient expression of the nuclease Cas9 through delivery of mRNA allowed for sufficient exposure to the nuclease for gene repair and reduced concerns associated with long-term nuclease exposure.

    “A key feature of our system is the transient expression of the nuclease Cas9, through delivery of mRNA. This allowed for sufficient exposure to the nuclease for gene repair and reduced concerns associated with long-term nuclease exposure34.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [34] Wu, X. et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014). source

      Not checked in the free run.

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

    Premisecitation

    CRISPR systems have potential advantages over ZFNs, most importantly their flexibility and ease in adjusting target site.

    “The CRISPR systems have a number of potential advantages over ZFNs, most importantly their flexibility and ease in adjusting target site4.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Mali, P., Esvelt, K.M. & Church, G.M. Cas9 as a versatile tool for engineering biology. Nat. Methods 10, 957–963 (2013). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    Correction of 6% of hepatocytes following a single round of treatment without selection suggests that this approach may be suitable for the treatment of a range of metabolic liver diseases and hemophilia, where restoration of 3-7% normal functional protein can be therapeutic.

    “However, correction of 6% of hepatocytes following a single round of treatment without selection suggests that this approach may be suitable for the treatment of a range of metabolic liver diseases and hemophilia, where restoration of 3–7% normal functional protein can be therapeutic39.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] Cox, D.B., Platt, R.J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21, 121–131 (2015). source

      Not checked in the free run.

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

    Interpretationcitation

    An alternative explanation for this observation is partial gene conversion in spite of 'G' and 'CC' single-nucleotide polymorphisms equidistant to the Cas9 cutting site.

    “An alternative explanation for this observation is partial gene conversion in spite of “G” and “CC” single-nucleotide polymorphisms equidistant to the Cas9 cutting site.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [36] Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J.A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cell. Biol. 18, 93–101 (1998). source

      Not checked in the free run.

    • cites [37] Goldberg, A.D. et al. Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691 (2010). source

      Not checked in the free run.

    • cites [38] Findlay, G.M., Boyle, E.A., Hause, R.J., Klein, J.C. & Shendure, J. Saturation editing of genomic regions by multiplex homology-directed repair. Nature 513, 120–123 (2014). source

      Not checked in the free run.

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

    Premisecitation

    Previous work has reported on the use of zinc finger nucleases (ZFNs) for in vivo gene correction.

    “Previous work has reported on the use of zinc finger nucleases (ZFNs) for in vivo gene correction31,33.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [31] Mahiny, A.J. et al. In vivo genome editing using nuclease-encoding mRNA corrects SP-B deficiency. Nat. Biotechnol. 33, 584–586 (2015). source

      Not checked in the free run.

    • cites [33] Li, H. et al. In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221 (2011). source

      Not checked in the free run.

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

    Interpretationown data

    It is possible that our stringent 'G-CC' pattern counting underestimated the HDR event.

    “It is possible that our stringent “G-CC” pattern counting underestimated the HDR event.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given

    Although NHEJ has been reported using AAV-mediated Cas9 delivery, HDR using viral delivery in vivo has not been reported.

    “Although NHEJ has been reported using AAV-mediated Cas9 delivery, HDR using viral delivery in vivo has not been reported.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Cells were tested by MIT core facility for mycoplasma contamination by an ELISA-based assay, and confirmed negative.

    “Cells were tested by MIT core facility for mycoplasma contamination by an ELISA-based assay, and confirmed negative.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Less than 10% indels at the Apob locus in the mouse liver was reported in the same study.

    “For example, less than 10% indels at the Apob locus in the mouse liver was reported in the same study12.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Ran, F.A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015). source

      Not checked in the free run.

40 of 40

  • The paper relies on this without support.

    Clinical implementation of CRISPR genome editing requires safe and effective delivery of all components into the nuclei of the target tissue.

    “However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue.”

  • The paper relies on this without support.

    Improvements to CRISPR delivery methods and HDR efficiency are required for therapeutic application of genome editing for disease gene correction.

    “However, improvements to CRISPR delivery methods and HDR efficiency are required for therapeutic application of genome editing for disease gene correction.”
2 of 2

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?

  • Gene-editing components can be delivered by lipid nanoparticle-mRNA formulations to treat genetic diseases, including hereditary tyrosinaemia type I, hypercholesterolaemia, lipoprotein metabolism disorders and transthyretin amyloidosis, as demonstrated in preclinical studies.

    “Gene-editing components can be delivered by lipid nanoparticle–mRNA formulations to treat genetic diseases, including HTI92,238, hypercholesterolaemia81,96,105,239, lipoprotein metabolism disorders85 and transthyretin amyloidosis239,240, which has been demonstrated in preclinical studies.”

    From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [92]

  • CRISPR can correct the Fah mutation through homology-directed repair or allelic exchange.

    “We and others recently reported that CRISPR can correct this Fah mutation through HDR16–18 or allelic exchange19.”

    From Adenine base editing in an adult mouse model of tyrosinaemia (2019) · cites this paper as [16]

  • In vitro Guide-Seq detected only the Fah target site and one off-target site (GOT1).

    “Using the standard Guide-seq protocol, we detected only the Fah target site and one off-target site (defined as Guide-seq off-target site 1, or GOT1) (Supplementary Data Set 1).”

    From Adenine base editing in an adult mouse model of tyrosinaemia (2019) · cites this paper as [16]

  • After a single treatment with C12-200 LNPs delivering Cas9 mRNA/sgRNA and AAV DNA donor, FAH expression in more than 6% of hepatocytes was repaired and symptoms of hepatic damage were prevented in the following 30 days in Fah knockout mice.

    “After a single treatment, the FAH expression in more than 6% of hepatocytes was repaired and the symptoms of hepatic damage were prevented in the following 30 days.”

    From RNA delivery biomaterials for the treatment of genetic and rare diseases (2019) · cites this paper as [141]

  • Yin et al.'s lipid nanoparticle-mediated delivery of Cas9 mRNA with AAV encoding sgRNAs and donor templates repaired FAH gene in a mouse model of human hereditary tyrosinemia, with correction efficiency >6% of hepatocytes after a single application and rescued disease symptoms.

    “Yin et al. combined lipid nanoparticle-mediated delivery of Cas9 mRNA with AAV encoding sgRNAs and donor templates to repair FAH gene in a mouse model of human hereditary tyrosinemia. The efficiency of correction was >6% of hepatocytes after a single application and the treatment rescued disease symptoms such as weight loss and liver damage [112].”

    From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [112]

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Authors
Yin, H., Song, C.-Q., Dorkin, J. R., Zhu, L. J., Li, Y., Wu, Q., Park, A., Yang, J., Suresh, S., Bizhanova, A., Gupta, A., Bolukbasi, M. F., Walsh, S., Bogorad, R. L., Gao, G., Weng, Z., Dong, Y., Koteliansky, V., Wolfe, S. A., Langer, R., Xue, W., & Anderson, D. G.
Affiliations
  • Koch Institute for Integrative Cancer Research At MIT (US)
  • University of Massachusetts Chan Medical School (US)
  • Tongji University (CN)
  • The Ohio State University (US)
  • Skolkovo Institute of Science and Technology (RU)
  • Harvard–MIT Division of Health Sciences and Technology (US)
Corresponding author
  • Xue, Wen
  • Anderson, Daniel G
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2016-02-01
Volume
34
Issue
3
Pages
328-333
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/nbt.3471
PMC
PMC5423356
OpenAlex
W2260024370
Cited by
876
References
47
Citations by year
2026: 26 · 2025: 56 · 2024: 44 · 2023: 52 · 2022: 90 · 2021: 108 · 2020: 110 · 2019: 104 · 2018: 128 · 2017: 112 · 2016: 46
FWCI
67.54
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion, cc-by)
  • PubMed Central (acceptedVersion, cc-by)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
  • eScholarship@UMassChan (submittedVersion)
Funders
  • National Institutes of Health (1f32eb017625-01)
  • National Eye Institute (P30EY012196)
  • National Cancer Institute (5‑U54‑CA151884‑04, U54CA151884, R00CA169512, P30CA014051, P30-CA-14051)
  • National Institute of Biomedical Imaging and Bioengineering (1F32EB017625, F32 EB017625)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
therapeutic genome editing, CRISPR/Cas9, lipid nanoparticle delivery, adeno-associated virus vectors, hereditary tyrosinemia, fumarylacetoacetate hydrolase
MeSH terms
Animals, Disease Models, Animal, Genetic Vectors, Humans, Lipids, Mutation, Viruses, Genome, Human, RNA Editing, Gene Transfer Techniques, Tyrosinemias, Mice, Nanoparticles, CRISPR-Cas Systems

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