Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1

Qing-Shuo Zhang, Amita Tiyaboonchai, Sean Nygaard, Kevin Baradar, Angela Major, Niveditha Balaji, Markus Grompe

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.

Original languageEnglish (US)
Pages (from-to)294-301
Number of pages8
JournalHuman Gene Therapy
Issue number5-6
StatePublished - Mar 2021


  • AAV
  • CRISPR/Cas9
  • Fanconi anemia
  • gene therapy

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics


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