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Cited by 200CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

Adenine base editing in an adult mouse model of tyrosinaemia

Open access
Song, Chun-Qing, Jiang, Tingting, Richter, Michelle, Rhym, Luke H., Koblan, Luke W., Zafra, Maria Paz, Schatoff, Emma M., Doman, Jordan L. +7 more·2019·Nature Biomedical Engineering
DOI 10.1038/s41551-019-0357-8PMC PMC6986236
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Abstract

In contrast to traditional CRISPR-Cas9 homology-directed repair, base editing can correct point mutations without supplying a DNA-repair template. Here we show in a mouse model of tyrosinaemia that hydrodynamic tail-vein injection of plasmid DNA encoding the adenine base editor (ABE) and a single-guide RNA (sgRNA) can correct an A>G splice-site mutation. ABE treatment partially restored splicing, generated fumarylacetoacetate hydrolase (FAH)-positive hepatocytes in the liver, and rescued weight loss in mice. We also generated FAH+ hepatocytes in the liver via lipid-nanoparticle-mediated delivery of a chemically modified sgRNA and an mRNA of a codon-optimized base editor that displayed higher base-editing efficiency than the standard ABEs. Our findings suggest that adenine base editing can be used for the correction of genetic diseases in adult animals.

AI snapshot

Written from the paper’s full text.

TL;DR

Hydrodynamic tail-vein delivery of an adenine base editor and sgRNA corrected an A>G splice-site mutation in Fahmut/mut mice, restoring splicing, generating Fah-positive hepatocytes, and rescuing weight loss. A codon-optimized RA6.3 improved editing, and lipid-nanoparticle delivery of RA6.3 mRNA/sgRNA also produced Fah-positive hepatocytes, though at low efficiency.

Key findings

  • ABE6.3 plus Fah sgRNA rescued weight loss in all five treated Fahmut/mut mice, whereas control PBS or ABE6.3 alone rapidly lost 20% of total body weight; two remaining ABE-treated mice were viable with normal body weight at 106 days without NTBC.
  • Amplicon deep sequencing showed A9-to-G correction of 9.5±4.0% (n=6), A6-to-G editing of 1.9±0.9%, and low indels (0.05% ABE vs 0.03% control); RT-PCR showed both 305 bp mutant and 405 bp wild-type-sized bands, and sequencing confirmed the corrected G at A9.
  • Codon-optimized RA6.3 increased A-to-G conversion in HEK293T cells by an average of 2.1±0.9 fold at site 1 and 4.8±1.8 fold at site 2; compared with HDR (site 1: 1.7±1.1%; site 2: 1.8±0.8%), ABE6.3 gave site 1: 30±6.3% and site 2: 7.3±1.6%, while RA6.3 gave site 1: 61±6.3% and site 2: 34±3.2%. In vivo, RA6.3 generated more Fah-positive hepatocytes than ABE6.3 (p<0.05).
  • LNP delivery of RA6.3 mRNA and chemically modified sgRNA produced 0.44%±0.28% edited hepatocytes, while control PBS showed none; Guide-Seq detected only the Fah target site and one off-target site (GOT1), with no increased A-to-G editing at GOT1 (n=4, p>0.05) or at four predicted top-ranking off-target sites (n=6, all rates <0.1%).

Methods

  • Proof-of-concept in vivo base editing in the Fahmut/mut hereditary tyrosinemia type I mouse model, using hydrodynamic tail-vein plasmid injection of ABE6.3 or RA6.3 plus Fah sgRNA, or LNP delivery of RA6.3 mRNA and modified sgRNA.
  • Mice were 8–10 week old Fahmut/mut mice kept on NTBC water; NTBC was withdrawn 6 days after hydrodynamic injection, and LNP-treated mice received 1 mg/kg RA6.3 mRNA plus 0.5 mg/kg Fah sgRNA in 3–4 doses every 3 days and were harvested 5 days after the last injection.
  • Analyses included Fah immunohistochemistry, RT-PCR spanning exons 5–9, amplicon deep sequencing for editing and indels, and Guide-Seq in Hepa1–6 cells followed by targeted deep sequencing in mouse livers.

Limitations

  • Stated: the in vivo ABE efficacy in this proof-of-concept study may not suffice for genetic disorders requiring higher correction and lacking selective proliferation of corrected cells; detecting off-target effects is difficult because on-target ABE efficiency is relatively low; LNP delivery was low efficiency, and ABE mRNA is longer than Cas9 mRNA (5.2 kb vs 4.1 kb), possibly reducing delivery or translation.
  • Evident from the text: sample sizes were small (n=5 for weight rescue, n=6 for editing/off-target sequencing, n=4 for GOT1), off-target assessment combined in vitro Guide-Seq with selected in vivo sites rather than comprehensive in vivo genome-wide profiling, and editing rates were low (A9 9.5±4.0%; LNP 0.44%±0.28%), limiting generalization.

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

Integrity signals

Registry

Crossref · 10.1038/s41551-019-0357-8

Cites

34 of 34 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 36 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    The A6 to G editing rate in livers was 1.9±0.9%.

    “We detected 1.9±0.9% A6 to G editing in livers (Fig. 2c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Fahmut/mut mice injected with control PBS or ABE6.3 alone rapidly lost 20% of total body weight.

    “As shown in Fig. 1c, Fahmut/mut mice injected with control PBS or ABE6.3 alone rapidly lost 20% of total body weight.”
    Quote in the paperSubstantial claim
  • Findingnone given

    The selected Fah sgRNA is the only sgRNA position holding the necessary NGG PAM and targeting the mutation A within the ABE editing window.

    “This selected sgRNA is the only sgRNA position holding the necessary “NGG” PAM and targeting the mutation “A” within the ABE editing window.”
    Quote in the paperSubstantial claim
  • Findingown data

    Sequencing of the 405 bp bands in ABE-treated mice confirmed the presence of the corrected G at position A9.

    “Sequencing of the 405 bp bands in ABE-treated mice confirmed the presence of the corrected “G” at position A9. (Fig. 2b).”
    Quote in the paperSubstantial claim
  • Findingown data

    In ABE-treated Fahmut/mut mice, both 305 and 405 bp PCR bands were observed, indicating that exon 8 to exon 9 splicing was restored in a subset of hepatocytes.

    “In ABE-treated Fahmut/mut mice (post NTBC withdrawal and hepatocyte expansion), we observed both 305 and 405 bp PCR bands, indicating that the exon 8 to exon 9 splicing was restored in a subset of hepatocytes.”
    Quote in the paperSubstantial claim
  • May combine two claims. 14
  • The quote only partly states this claim. 1
  • Classified as probably not a substantial claim. 8

  • May combine two claims.

    Findingown data

    Wild-type liver had a 405 bp PCR band containing exon 8 and Fahmut/mut liver had a 305 bp PCR band corresponding with truncated Fah mRNA lacking exon 8.

    “As shown in Fig. 2a, we found that wild-type liver had a 405 bp PCR band containing exon 8 and Fahmut/mut liver had a 305 bp PCR band corresponding with truncated Fah mRNA lacking exon 8.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    The rate of both A6 to G and A9 to G editing is low (~0.1%).

    “The rate of both A6 to G and A9 to G editing (Fig. 2c) is low (~0.1%).”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    No A•T to G•C editing above background was detected at four predicted top-ranking off-target sites in ABE-treated livers (n=6; all rates <0.1%).

    “To expand our analysis, we sequenced four predicted top-ranking off-target sites in ABE-treated livers (n=6), and did not detect A•T to G•C editing above the background level in a control liver (all rates are <0.1%) (Supplementary Fig. 6).”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Adenine base editors comprise an evolved Escherichia coli TadA and Cas9 nickase and mediate A•T to G•C substitutions.

    “Adenine BEs comprise an evolved Escherichia coli TadA (ecTadA*, a tRNA adenosine deaminase evolved to accept DNA substrate) and Cas9 nickase, and mediate A•T to G•C substitutions1.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [1] Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). source

      Not checked in the free run.

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

    Premisecitation

    Local delivery of ABE by intramuscular injection of trans-splicing AAV has been reported.

    “Local delivery of ABE by intramuscular injection of trans-splicing AAV was recently reported12; however, systemic delivery of ABE for correction of liver disease in adult animals has not been investigated.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Ryu, S. M. et al. Adenine base editing in mouse embryos and an adult mouse model of Duchenne muscular dystrophy. Nat. Biotechnol. 36, 536–539 (2017). source

      Not checked in the free run.

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

    Premisecitation

    Point mutations are a frequent cause of genetic disease, and approximately 50% of disease point mutations are G:C>A:T.

    “Point mutations are a frequent cause of genetic disease, with ~50% of disease point mutations being G:C>A:T 1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017). source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    FAH deficiency leads to accumulation of toxic metabolic intermediates, causing apoptosis of mutant hepatocytes and severe liver damage.

    “FAH deficiency leads to accumulation of toxic metabolic intermediates, causing apoptosis of mutant hepatocytes and severe liver damage.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Azuma, H. et al. Robust expansion of human hepatocytes in Fah −/−/Rag2 −/−/Il2rg −/− mice. Nat. Biotechnol. 25, 903–910 (2007). source

      Not checked in the free run.

    • cites [14] 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

    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.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [16] Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328–333 (2016). source

      Not checked in the free run.

    • cites [17] Song, C. Q. & Xue, W. CRISPR–Cas-related technologies in basic and translational liver research. Nat. Rev. Gastroenterol. Hepatol. 15, 251–252 (2018). source

      Not checked in the free run.

    • cites [18] Shao, Y. et al. Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats. J. Biol. Chem. 293, 6883–6892 (2018). source

      Not checked in the free run.

    • cites [19] Wang, D. et al. Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice. Nat. Biotechnol. 36, 839–842 (2018). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Base editing enables precise base changes.

    “A new-generation of genome editing tool, Base Editing (BE) system, enables precise base changes (reviewed in 4, 5), does not induce a high-level of DSBs, and finally does not require HDR donors for gene repair 4, 5.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [4] Hess, G. T., Tycko, J., Yao, D. & Bassik, M. C. Methods and applications of CRISPR-mediated base editing in eukaryotic genomes. Mol. Cell 68, 26–43 (2017). source

      Not checked in the free run.

    • cites [5] Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet.19, 770–788 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Transient delivery of ABE and sgRNA by non-viral hydrodynamic injection is sufficient to generate Fah+ hepatocytes and rescue the phenotypic weight loss of Fah mutant mice.

    “In summary, our results demonstrate that transient delivery of ABE and sgRNA by non-viral hydrodynamic injection is sufficient to generate Fah+ hepatocytes and rescue the phenotypic weight loss of Fah mutant mice.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Optimized cytosine BEs and ABE7.10 have been reported.

    “Optimized cytosine BEs and ABE7.10 have been reported 26, 27 but ABE6.3 has not been developed.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [26] Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018). source

      Not checked in the free run.

    • cites [27] Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    ABE can directly correct the Fah mutation without causing a high level of indel mutations.

    “These data suggest that ABE can directly correct the Fah mutation without causing a high level of indel mutations.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Efficiency of base editing can be improved with codon optimization of the Cas9 nickase and inclusion of an N-terminal nuclear localization sequence (NLS).

    “New reports have indicated that efficiency of base editing can be improved with codon optimization of the Cas9 nickase and inclusion of an N-terminal nuclear localization sequence (NLS) 26, 27.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [26] Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36, 888–893 (2018). source

      Not checked in the free run.

    • cites [27] Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843–846 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In vivo delivery of ABE was able to correct the Fah mutation in vivo without inducing high-level indels.

    “We reported the first codon-optimized ABE6.3 and showed that in vivo delivery of ABE was able to correct the Fah mutation in vivo without inducing high-level indels.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All animal study protocols were approved by the UMass IACUC.

    “All animal study protocols were approved by the UMass IACUC.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Mice with more than 20% weight loss were humanely euthanized according to guidelines.

    “Mice with more than 20% weight loss were humanely euthanized according to guidelines.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    ABE-treated hepatocytes have long-term viability and functionality.

    “Two remaining ABE-treated mice were viable with normal body weight at 106 days without NTBC treatment (Fig. 2d and Supplementary Fig. 4), demonstrating the long-term viability and functionality of ABE treated hepatocytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    RA6.3 is a robust base editor in mammalian cells and in the mouse liver.

    “Our combined in vitro and in vivo results indicate that RA6.3 is a robust base editor in mammalian cells and in the mouse liver.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Because S235 is near the active site of the Fah enzyme, the A6G/A9G allele may compromise enzyme activity and impede functional rescue of edited hepatocytes.

    “Because S235 is near the active site of the Fah enzyme22, the A6G/A9G allele may compromise enzyme activity and impede functional rescue of edited hepatocytes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [22] Bateman, R. L. et al. Mechanistic inferences from the crystal structure of fumarylacetoacetate hydrolase with a bound phosphorus-based inhibitor. J. Biol. Chem. 276, 15284–15291 (2001). source

      Not checked in the free run.

19 of 19

  • The paper relies on this without support.

    Systemic delivery of ABE for correction of liver disease in adult animals has not been investigated.

    “Local delivery of ABE by intramuscular injection of trans-splicing AAV was recently reported12; however, systemic delivery of ABE for correction of liver disease in adult animals has not been investigated.”

  • The paper relies on this without support.

    ABE6.3 has not been developed as a codon-optimized editor.

    “Optimized cytosine BEs and ABE7.10 have been reported 26, 27 but ABE6.3 has not been developed.”

  • The paper relies on this without support.

    ABE mRNA is longer than Cas9 mRNA (5.2 kb vs 4.1 kb).

    “Because ABE mRNA is longer than Cas9 mRNA (5.2 kb vs 4.1 kb), it is possible that the delivery or translation of ABE mRNA is less efficient than Cas9 mRNA.”
3 of 3

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?

  • Ionizable lipid nanoparticles (LNPs) are a promising non-viral delivery platform for mRNA nanomedicines and have been used to deliver siRNA and CRISPR-based gene editing therapies to the liver in postnatal mouse, non-human primate, and clinical studies with encouraging results.

    “Ionizable lipid nanoparticles (LNPs) are a promising non-viral delivery platform for mRNA nanomedicines (13, 14) that have been used to deliver siRNA and CRISPR-based gene editing therapies to the liver in postnatal mouse, non-human primate, and clinical studies with encouraging results (15–18).”

    From Ionizable Lipid Nanoparticles for Therapeutic Base Editing of Congenital Brain Disease (2023) · cites this paper as [15]

    Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • 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 [238]

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Authors
Song, C.-Q., Jiang, T., Richter, M., Rhym, L. H., Koblan, L. W., Zafra, M. P., Schatoff, E. M., Doman, J. L., Cao, Y., Dow, L. E., Zhu, L. J., Anderson, D. G., Liu, D. R., Yin, H., & Xue, W.
Affiliations
  • University of Massachusetts Chan Medical School (US)
  • Broad Institute (US)
  • Koch Institute for Integrative Cancer Research At MIT (US)
  • Weill Cornell Medicine (US)
  • Wuhan University (CN)
Corresponding author
  • Liu, David R.
  • Yin, Hao
  • Xue, Wen
ORCID
Venue
Nature Biomedical Engineering
Publisher
Springer Science and Business Media LLC
Published
2019-02-25
Volume
4
Issue
1
Pages
125-130
ISSN
2157-846X (ISSN-L)
DOI
10.1038/s41551-019-0357-8
PMC
PMC6986236
OpenAlex
W2918145920
Cited by
200
References
34
Citations by year
2026: 20 · 2025: 20 · 2024: 28 · 2023: 27 · 2022: 27 · 2021: 33 · 2020: 39 · 2019: 4 · 2018: 2
FWCI
8.52
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biomedical Engineering (publishedVersion)
  • PubMed (publishedVersion)
  • Institutional Repository of the University of Granada (University of Granada) (publishedVersion, other-oa)
  • PubMed Central (acceptedVersion)
  • Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion)
  • The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion)
Funders
  • National Heart, Lung, and Blood Institute (P01HL131471, DP2HL137167, UG3HL147367)
  • National Human Genome Research Institute (RM1HG009490)
  • National Cancer Institute (K22CA181280, R01CA195787, T32CA203702, F31CA224800, P30CA014051)
  • National Institute of General Medical Sciences (T32GM095450, R35GM118062)
  • National Institute of Allergy and Infectious Diseases (U01AI142756)
  • National Institute of Biomedical Imaging and Bioengineering (R01EB022376)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
adenine base editing, tyrosinaemia, fumarylacetoacetate hydrolase, splice site mutation, single guide RNA, hydrodynamic injection, lipid nanoparticle delivery, hepatocytes, weight loss
MeSH terms
Gene Editing, Adenine, Animals, Disease Models, Animal, Female, Humans, Hydrolases, Liver, RNA, Point Mutation, Tyrosinemias, Hepatocytes, HEK293 Cells

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