Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype
Open accessAbstract
We demonstrate CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ∼1/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9–mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
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Written from the paper’s full text.
TL;DR
CRISPR-Cas9 plus a single-stranded DNA donor delivered by hydrodynamic injection corrected a Fah splicing mutation in adult Fah^mut/mut mice, restoring wild-type Fah expression and rescuing the body-weight-loss phenotype. The study supports proof-of-principle in vivo genome editing in adult liver, while noting delivery and efficiency barriers to clinical translation.
Key findings
- Initial Fah repair frequency in hepatocytes was 0.40 ± 0.12% (n = 3 mice) with FAH2 sgRNA versus 0.01 ± 0.02% with unguided Cas9, corresponding to wild-type Fah protein in ~1/250 liver cells.
- After positive selection, FAH2-treated Fah^mut/mut mice had widespread Fah+ hepatocyte patches at 33.5% ± 3.3% (n = 3 mice) after 33 days and 30 days off NTBC; Fah mRNA reached 8–36% of wild-type levels, and deep sequencing detected ~9% A→G correction (n = 2) with ~26% indels.
- Treated mice did not lose weight or lost <20% after 30 days without NTBC, and FAH2-treated mice showed less liver damage by histology and serum AST, ALT, and bilirubin; RT-PCR showed restoration of exon 8–9 splicing.
- No cleavage was detected at three to four top-ranking predicted off-target sites per sgRNA, and <0.3% indels were detected at three sequenced FAH2 off-target sites.
Methods
- Adult Fah^mut/mut mice, a model of hereditary tyrosinemia type I carrying the same homozygous G→A mutation as human disease, were given hydrodynamic tail vein injections of saline, ssDNA donor alone, ssDNA plus unguided Cas9, or ssDNA plus Cas9 with one of three Fah sgRNAs (FAH1–3).
- A 199-nt single-stranded DNA donor carried the wild-type G nucleotide and homology arms flanking the sgRNA target.
- Outcomes were assessed by body weight, liver histology, serum AST/ALT/bilirubin, Fah immunohistochemistry, RT-PCR and quantitative RT-PCR, deep sequencing, and Surveyor nuclease off-target assays in mouse 3T3 cells.
Limitations
- The paper states that hydrodynamic injection is unlikely to be used clinically and that improvements in CRISPR delivery and repair efficiency are needed for broad therapeutic application, especially to increase gene correction and target other tissues.
- The paper states that further studies are required to evaluate off-target effects, particularly in vivo, and notes that an indel rate of ~26% was detected, with further work needed to assess initial indel formation versus gene correction.
- The paper notes deep sequencing could not detect low-frequency single-nucleotide polymorphisms in hepatocytes because of sequencing error, nonparenchymal cell mixture, and hepatocyte polyploidy; evident from the text, the in vivo correction experiments used small numbers of mice (n = 2–3) and relied on the strong positive selection of corrected Fah+ hepatocytes in this disease model.
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Claims the paper makes
0 of 16 cited sources checked · 0 supported · 0 flagged
- Findingown data
CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes was achieved in a mouse model of hereditary tyrosinemia type I.
“We demonstrate CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia.”
Quote in the paperSubstantial claim - Findingown data
An indel rate of approximately 26% was detected by sequencing in FAH2-treated livers at 30 days off NTBC.
“an indel rate of ~26% was also detected by sequencing of these samples”
Quote in the paperNot a substantial claim - Findingown data
FAH1- or FAH3-treated mice regained all the weight they had lost after a second round of NTBC withdrawal.
“these mice regained all the weight they had lost”
Quote in the paperSubstantial claim - Findingown data
The A→G correction rate in FAH2-treated livers at 30 days off NTBC was approximately 9% (n = 2) as detected by deep sequencing.
“the A→G correction rate (~9%, n = 2) detected by deep sequencing in FAH2-treated livers at 30 d off NTBC”
Quote in the paperSubstantial claim - Findingown data
Hydrodynamic injection of CRISPR-Cas9 components resulted in initial expression of wild-type Fah protein in approximately 1/250 liver cells (0.40 ± 0.12%).
“Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in ~1/250 liver cells.”
- Also stated in: § Abstract · plain
“the initial repair frequency was 0.40 ± 0.12% (n = 3 mice) for mice treated with FAH2 compared to 0.01 ± 0.02% for those with unguided Cas9”
Quote in the paperSubstantial claim - Also stated in: § Abstract · plain
- Findingown data
Weight loss in FAH1- and FAH3-treated mice was less than 20% after 30 days without NTBC water.
“weight loss in FAH1- and FAH3-treated mice was <20% after 30 d without NTBC water”
Quote in the paperSubstantial claim - Findingown data
CRISPR-Cas9-treated mice had Fah mRNA at 8-36% of wild-type levels at the end of NTBC water withdrawal.
“Quantitative RT-PCR using primers spanning exons 8 to 9 on liver samples from CRISPR-Cas9–treated mice showed they had Fah mRNA at 8–36% of levels in wild-type mice at the end of NTBC water withdrawal”
Quote in the paperSubstantial claim - Findingown data
Cleavage was not detected at the assayed three to four top-ranking off-target sites for each sgRNA.
“Cleavage was not detected at the assayed three to four top-ranking off-target sites for each sgRNA”
Quote in the paperSubstantial claim - Findingown data
Exon 8 to exon 9 splicing of Fah is restored in a subset of hepatocytes in Fahmut/mut mice injected with FAH1, FAH2, or FAH3.
“Fahmut/mut mice injected with FAH1, 2 or 3 had both the 305- and 405-bp PCR bands, indicating that the exon 8 to exon 9 splicing is restored in a subset of hepatocytes”
Quote in the paperSubstantial claim - Interpretationown data
The study demonstrates the potential to correct disease genes in vivo in adult mouse liver using a CRISPR-Cas9 system.
“these data demonstrate the potential to correct disease genes in vivo in adult mouse liver using a CRISPR-Cas9 system.”
Quote in the paperSubstantial claim - Interpretationown data
Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype in Fahmut/mut mice.
“Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype.”
Quote in the paperSubstantial claim - Interpretationown data
The reduction in liver damage in FAH2-treated mice suggests a functional rescue of the Fah deficiency-induced liver damage.
“suggesting a functional rescue of the Fah deficiency– induced liver damage.”
Quote in the paperSubstantial claim - Premisecitation
Repair of 1/10,000 hepatocytes was reported to rescue the phenotype of Fahmut/mut mice.
“repair of 1/10,000 hepatocytes was reported to rescue the phenotype of Fahmut/mut mice8.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [8] Paulk, N.K. et al. Hepatology 51, 1200–1208 (2010). source
Not checked in the free run.
- Interpretationown data
Transient expression of Cas9, sgRNA, and a co-injected ssDNA by non-viral hydrodynamic injection is sufficient to restore the weight loss of a mouse model of hereditary tyrosinemia type I.
“Transient expression of Cas9, sgRNA and a co-injected ssDNA by non-viral hydrodynamic injection is sufficient to restore the weight loss of a mouse model of HTI.”
Quote in the paperSubstantial claim - Interpretationcitation
The strong positive selection and expansion of Fah+ hepatocytes in the Fahmut/mut liver may have contributed to the correction of the disease phenotype, given the observed initial genetic correction rate of approximately 1/250 cells.
“The strong positive selection and expansion of Fah+ hepatocytes in the Fahmut/mut liver may have contributed to the correction of the disease phenotype8, given the observed initial genetic correction rate of ~1/250 cells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [8] Paulk, N.K. et al. Hepatology 51, 1200–1208 (2010). source
Not checked in the free run.
- Study design and conductcitation
Deep sequencing could not detect low-frequency single-nucleotide polymorphisms in hepatocytes due to sequencing error rate, mixture of nonparenchymal cells, and polyploidy of hepatocytes.
“due to the error rate of sequencing, the mixture of nonparenchymal cells and polyploidy of hepatocytes, this approach could not detect low-frequency single-nucleotide polymorphisms in hepatocytes11.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [11] Nielsen, R., Korneliussen, T., Albrechtsen, A., Li, Y. & Wang, J. PLoS ONE 7, e37558 (2012). source
Not checked in the free run.
- Interpretationown data
Correction of genetic disease in vivo with CRISPR-Cas9 may be possible.
“This proof-of-principle study indicates that correction of genetic disease in vivo with CRISPR-Cas9 may be possible”
Quote in the paperSubstantial claim - Interpretationown data
CRISPR-Cas9-mediated genome editing has potential for correction of human genetic diseases.
“has potential for correction of human genetic diseases.”
Quote in the paperSubstantial claim - Interpretationown data
CRISPR-Cas9-mediated genome editing is possible in adult animals.
“Our study indicates that CRISPR-Cas9–mediated genome editing is possible in adult animals”
Quote in the paperSubstantial claim - Premisecitation
FAH deficiency causes accumulation of toxic metabolites, such as fumarylacetoacetate, in hepatocytes, resulting in severe liver damage.
“FAH deficiency causes accumulation of toxic metabolites, such as fumarylacetoacetate, in hepatocytes, resulting in severe liver damage8.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [8] Paulk, N.K. et al. Hepatology 51, 1200–1208 (2010). source
Not checked in the free run.
- Interpretationown data
Hydrodynamic injection of Cas9 plus sgRNA components was well-tolerated in wild-type FVB mice.
“suggesting that hydrodynamic injection of these components was well-tolerated.”
Quote in the paperSubstantial claim - Premisecitation
The Fah5981SB mouse model harbors the same homozygous G→A point mutation of the last nucleotide of exon 8 as causes the human disease.
“The Fah5981SB mouse model8,9 (referred to here as Fahmut/mut) of HTI harbors the same homozygous G→A point mutation of the last nucleotide of exon 8 as causes the human disease.”
Quote in the paperSubstantial claim0 of 2 sources checked
- The quote only partly states this claim. 2
- May combine two claims. 8
- Classified as probably not a substantial claim. 6
The quote only partly states this claim.
Findingown dataMice receiving oligo plus pX330 expressing Cas9 plus FAH2 did not lose weight after 30 days without NTBC water.
“Mice receiving oligo plus pX330 expressing Cas9 plus FAH2 did not lose weight”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataFahmut/mut mice injected with saline, ssDNA oligo alone, or unguided Cas9 and kept without NTBC-containing water rapidly lost 20% of their body weight and had to be euthanized.
“Fahmut/mut mice injected with saline, ssDNA oligo alone or unguided Cas9, and kept without NTBC-containing water, rapidly lost 20% of their body weight and had to be euthanized”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataCleavage was detected at Fah in 3T3 cells transfected with FAH1, FAH2, or FAH3, indicating that the one nucleotide mismatch between these sgRNAs and the wild-type Fah gene does not prevent Cas9-mediated editing.
“Cleavage was detected at Fah in 3T3 cells, indicating that the one nucleotide mismatch between FAH1, 2 and 3 and the wild-type Fah gene does not prevent Cas9-mediated editing”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataWild-type FVB mice injected with Cas9 plus sgRNA showed no body weight loss, no signs of hyperplasia, and extremely low Cas9 expression after 3 months.
“Wildtype FVB mice injected with Cas9 plus sgRNA showed no body weight loss, no signs of hyperplasia and extremely low Cas9 expression after 3 months”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataThirty-three days after FAH2 treatment and 30 days without NTBC water, Fahmut/mut mice had widespread patches of Fah+ hepatocytes (33.5% ± 3.3%, n = 3 mice).
“sites Fahmut/mut mice had widespread patches of Fah+ hepatocytes (33.5% ± 3.3%, n = 3 mice)”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationCRISPR-Cas9 genome editing has been applied to correct disease-causing mutations in mouse zygotes and human cell lines for cataract and cystic fibrosis.
“CRISPR-Cas9 genome editing has been applied to correct disease-causing mutations in mouse zygotes and human cell lines for cataract5 and cystic fibrosis6”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTargeted integration by homologous recombination of adeno-associated virus carrying wild-type Fah sequence could achieve stable gene repair in vivo in mice, but required multiple rounds of NTBC withdrawal and recovery.
“A previous study showed that targeted integration by homologous recombination of adeno-associated virus carrying the wild-type Fah sequence could achieve stable gene repair in vivo in mice, but required multiple rounds of NTBC withdrawal and recovery8.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [8] Paulk, N.K. et al. Hepatology 51, 1200–1208 (2010). source
Not checked in the free run.
May combine two claims.
PremisecitationLiver cells in which Fah has been repaired have a selective advantage and can expand and repopulate the liver.
“Liver cells in which Fah has been repaired have a selective advantage and can expand and repopulate the liver8.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [8] Paulk, N.K. et al. Hepatology 51, 1200–1208 (2010). source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable).
InterpretationcitationDiseases in which positive selection of corrected cells occurs may be particularly suitable for gene repair-based therapy.
“Diseases in which positive selection of corrected cells occurs8,9 may be particularly suitable for gene repair–based therapy”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationThe initial efficiency of repair using zinc finger nucleases was low and was substantially improved by subsequent work to correct hemophilia in mice.
“the initial efficiency of repair using zinc finger nucleases was also low and was substantially improved by subsequent work to correct hemophilia in mice12.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [12] Li, H. et al. Nature 475, 217–221 (2011). source
Not checked in the free run.
Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based).
Interpretationnone givenHydrodynamic injection is unlikely to be used for clinical implementation.
“hydrodynamic injection is unlikely to be used for clinical implementation.”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationThe Fah mutation causes skipping of exon 8 during splicing and formation of truncated, unstable FAH protein.
“This mutation causes skipping of exon 8 during splicing and formation of truncated, unstable FAH protein (Fig. 1a).”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S2 (checkable).
InterpretationcitationCRISPR-Cas9-mediated correction may be translatable to humans.
“we believe that recent advances in the delivery of nucleic acid therapeutics provide hope that CRISPR-Cas9–mediated correction may be translatable to humans15.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [15] Kanasty, R., Dorkin, J.R., Vegas, A. & Anderson, D. Nat. Mater. 12, 967–977 (2013). source
Not checked in the free run.
The paper relies on this without support.
Delivery to adult mammalian organs to correct genetic disease genes had not been reported.
“but delivery to adult mammalian organs to correct genetic disease genes has not been reported to our knowledge.”
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?
Hydrodynamic injection of CRISPR-Cas9 DNA and a short single-stranded DNA HDR template can correct the Fah mutation in the mouse liver.
“Previously, our groups have shown that hydrodynamic injection of CRISPR-Cas9 DNA and a short single-stranded DNA HDR template can correct the Fah mutation in the mouse liver16.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [16]
CRISPR/Cas9 system holds tremendous promise for gene therapy and can correct causal mutations in the original genome and rescue disease phenotypes of monogenic disorders permanently.
“Furthermore, CRISPR/Cas9 system holds tremendous promise for gene therapy [46–51]. It can correct causal mutations in the original genome and rescue the disease phenotypes of monogenic disorders permanently, which currently represents the most translatable field in CRISPR/Cas9-mediated disease therapy.”
From Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018) · cites this paper as [46]
Yin et al delivered CRISPR-Cas9 and a donor template into adult mice with hereditary tyrosinemia via tail-vein hydrodynamic injection, resulting in gene corrections in 0.25% of liver cells initially and 33.5% at 33 days, sufficient to rescue the disease phenotype.
“Yin et al, 32 for example, delivered CRISPR-Cas9 agents and a homologous donor template (in order to increase HDR rate) into adult mice with hereditary tyrosinemia via tail-vein hydrodynamic injection, resulting in gene corrections in 0.25% of liver cells initially, and in 33.5% of liver cells 33 days postinjection ( possibly due to selective advantages imposed by the correction), which was suffic”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [32]
This method is more translatable to human therapeutics because it does not involve embryo manipulations.
“This method is more translatable to human therapeutics because it does not involve embryo manipulations.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [32]
CRISPR-Cas9 can mediate somatic gene corrections in adult animals, bypassing embryo manipulations.
“CRISPR-Cas9 technology can also mediate somatic gene corrections in adult animals, bypassing embryo manipulations.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [32]
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- Authors
- Yin, H., Xue, W., Chen, S., Bogorad, R. L., Benedetti, E., Grompe, M., Koteliansky, V., Sharp, P. A., Jacks, T., & Anderson, D. G.
- Affiliations
- Koch Institute for Integrative Cancer Research At MIT (US)
- Oregon Health & Science University (US)
- Skolkovo Institute of Science and Technology (RU)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Anderson, Daniel G
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-03-30
- Volume
- 32
- Issue
- 6
- Pages
- 551-553
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4157757
- OpenAlex
- W1504222414
- Semantic Scholar
- 83863be61c174889ac0365f3b3cc9929e92f2312
- Cited by
- 951
- References
- 15
- Citations by year
- 2026: 17 · 2025: 33 · 2024: 24 · 2023: 62 · 2022: 52 · 2021: 65 · 2020: 77 · 2019: 88 · 2018: 109 · 2017: 112 · 2016: 129 · 2015: 120 · 2014: 62 · 2013: 1
- FWCI
- 57.77
- Type
- journal article
- Language
- en
- Update notices
- erratum (2014-09-09)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- PMC
- Funders
- Howard Hughes Medical Institute
- National Cancer Institute (K99CA169512, 5‑U54‑CA151884‑04, R01CA133404, U54CA151884, P01CA042063, R00CA169512, 2-PO1-CA42063, P30CA014051, P30-CA-14051)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK048252)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, genome editing, hereditary tyrosinemia, fumarylacetoacetate hydrolase, hydrodynamic gene delivery, hepatocytes, liver, adult mice, weight loss
- MeSH terms
- Gene Editing, Animals, Hydrolases, Mice, Transgenic, Mutation, Phenotype, Genetic Therapy, Genome, Treatment Outcome, Tyrosinemias, Mice, CRISPR-Cas Systems, CRISPR-Associated Proteins
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