CRISPR/Cas9 - Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

EBioMedicine. 2017 Jun;20:19-26. doi: 10.1016/j.ebiom.2017.05.015. Epub 2017 May 11.

Abstract

Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR)-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ)-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold) than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah-/- liver failure mice, offering an efficient approach for precisely targeted gene therapies.

Keywords: CRISPR/Cas9; Fah(−/−) mice; Gene therapy; In vivo targeted integration; MMEJ.

MeSH terms

  • Animals
  • Biomarkers
  • CRISPR-Cas Systems*
  • Cell Line
  • Female
  • Gene Targeting*
  • Gene Transfer Techniques
  • Genetic Engineering
  • Genetic Therapy
  • Genetic Vectors / genetics
  • Genotype
  • Hepatocytes / metabolism
  • Humans
  • Hydrolases / genetics
  • Male
  • Mice
  • Mice, Knockout
  • Neurons / metabolism
  • Phenotype
  • Recombinational DNA Repair*

Substances

  • Biomarkers
  • Hydrolases
  • fumarylacetoacetase