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Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction

Gene Therapy volume 30, pages 386–397 (2023)Cite this article

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Abstract

Gene editing for the cure of inborn errors of metabolism (IEMs) has been limited by inefficiency of adult hepatocyte targeting. Here, we demonstrate that in utero CRISPR/Cas9-mediated gene editing in a mouse model of hereditary tyrosinemia type 1 provides stable cure of the disease. Following this, we performed an extensive gene expression analysis to explore the inherent characteristics of fetal/neonatal hepatocytes that make them more susceptible to efficient gene editing than adult hepatocytes. We showed that fetal and neonatal livers are comprised of proliferative hepatocytes with abundant expression of genes involved in homology-directed repair (HDR) of DNA double-strand breaks (DSBs), key for efficient gene editing by CRISPR/Cas9. We demonstrated the same is true of hepatocytes after undergoing a regenerative stimulus (partial hepatectomy), where post-hepatectomy cells show a higher efficiency of HDR and correction. Specifically, we demonstrated that HDR-related genome correction is most effective in the replicative phase, or S-phase, of an actively proliferating cell. In conclusion, this study shows that taking advantage of or triggering cell proliferation, specifically DNA replication in S-phase, may serve as an important tool to improve efficiency of CRISPR/Cas9-mediated genome editing in the liver and provide a curative therapy for IEMs in both children and adults.

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Fig. 1: AAV-HT and AAV-Cas9 administration to mice.
Fig. 2: Expression of HDR-related genes and efficiency of CRISPR/Cas9-mediated gene editing in fetal, neonatal, and adult hepatocytes.
Fig. 3: Cell cycle synchronization and efficiency of CRISPR/Cas9-mediated gene editing.
Fig. 4: Expression of HDR-related genes and efficiency of CRISPR/Cas9-mediated gene editing in adult hepatocytes before and after regenerative stimulus.

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Data availability

Sanger sequencing data is being uploaded to a public online repository and accession numbers will be provided. The remainder of data are available in the main text and supplementary materials.

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Acknowledgements

We thank Kari Allen for help with animal care. We thank Raymond Hickey for help with initial experiments. We thank LouAnn Gross and Tony Blahnik for histology and immunohistochemistry support. Children’s Hospital of Minnesota Foundation.

Author information

Author notes

  1. These authors contributed equally: Gourish Mondal, Caitlin J. VanLith.

Authors and Affiliations

  1. Department of Surgery, Mayo Clinic, Rochester, MN, USA

    Gourish Mondal, Caitlin J. VanLith, Clara T. Nicolas, William S. Cao, Lori Hillin, Benjamin J. Haugo, Robert A. Kaiser & Joseph B. Lillegard

  2. Department of Surgery, University of Alabama Birmingham, Birmingham, AL, USA

    Clara T. Nicolas

  3. Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA

    Whitney S. Thompson

  4. Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA

    Daniel R. O’ Brien & Jean-Pierre Kocher

  5. Midwest Fetal Care Center, Children’s Hospital of Minnesota, Minneapolis, MN, USA

    Robert A. Kaiser & Joseph B. Lillegard

  6. Pediatric Surgical Associates, Minneapolis, MN, USA

    Joseph B. Lillegard

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Contributions

Conceptualization: JBL, GM, and CTN. Methodology: JBL, GM, and CTN. Validation: RAK. Formal Analysis: CJV, GM, and DRO. Investigation: GM, CVL, and CTN. Resources: LH, WC, and BH. Writing-Original Draft: GM, CJV, CTN, and WST. Writing-Review & Editing: JBL and CTN. Visualization: CTN and JBL. Supervision: JBL and RAK. Project administration: RAK. Funding Acquisition: JBL.

Corresponding author

Correspondence to Joseph B. Lillegard.

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Competing interests

JBL is Chief Scientific Officer and RAK is Vice-President of Pre-Clinical Development of Castle Creek Biosciences, Inc. No funding or financial support for this work has been provided by Castle Creek Biosciences. The remaining authors have no competing interests.

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All animal experiments conducted in this study were approved by Mayo Clinic’s Institutional Animal Care and Use Committee.

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Mondal, G., VanLith, C.J., Nicolas, C.T. et al. Activation of homology-directed DNA repair plays key role in CRISPR-mediated genome correction. Gene Ther 30, 386–397 (2023). https://doi.org/10.1038/s41434-022-00369-8

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