AAV-Mediated Somatic Gene Editing for Cardiac and Skeletal Muscle in a Large Animal Model

Methods Mol Biol. 2022:2573:63-74. doi: 10.1007/978-1-0716-2707-5_6.

Abstract

Here we describe a protocol to produce a recombinant adeno-associated viral vector (rAAV)-based system to deliver the CRISPR-Cas9 complex into porcine skeletal muscle and myocardial cells. We initially describe the genomic composition of the rAAV-CRISPR vectors used in our lab. Furthermore, we give a step-by-step instruction into the production of recombinant viral vectors with high yields and purity. Lastly we describe the minimally invasive injection regimes to target the myocardium in a pig.

Keywords: CRISPR; Cas9; Duchenne muscular dystrophy; Gene editing; Gene vector; rAAV.

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Dependovirus / genetics
  • Dependovirus / metabolism
  • Disease Models, Animal
  • Dystrophin / genetics
  • Gene Editing* / methods
  • Genetic Therapy / methods
  • Genetic Vectors / genetics
  • Muscle, Skeletal / metabolism
  • Muscular Dystrophy, Duchenne* / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Swine

Substances

  • Dystrophin
  • RNA, Guide, CRISPR-Cas Systems