Precise in vivo genome editing via single homology arm donor mediated intron-targeting gene integration for genetic disease correction

Cell Res. 2019 Oct;29(10):804-819. doi: 10.1038/s41422-019-0213-0. Epub 2019 Aug 23.

Abstract

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. As a proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • DNA Repair
  • Dependovirus / genetics
  • GATA3 Transcription Factor / genetics
  • Gene Editing / methods*
  • Gene Knock-In Techniques
  • Genetic Therapy / methods
  • Genetic Vectors / metabolism
  • Human Embryonic Stem Cells
  • Humans
  • Introns
  • Mice
  • Mice, Inbred C57BL
  • Mice, Inbred ICR
  • Neurons / cytology
  • Neurons / metabolism
  • RNA, Guide / metabolism
  • Rats
  • Tubulin / genetics

Substances

  • GATA3 Transcription Factor
  • RNA, Guide
  • Tubb3 protein, rat
  • Tubulin