In Vivo Ryr2 Editing Corrects Catecholaminergic Polymorphic Ventricular Tachycardia

Circ Res. 2018 Sep 28;123(8):953-963. doi: 10.1161/CIRCRESAHA.118.313369.


Rationale: Autosomal-dominant mutations in ryanodine receptor type 2 ( RYR2) are responsible for ≈60% of all catecholaminergic polymorphic ventricular tachycardia. Dysfunctional RyR2 subunits trigger inappropriate calcium leak from the tetrameric channel resulting in potentially lethal ventricular tachycardia. In vivo CRISPR/Cas9-mediated gene editing is a promising strategy that could be used to eliminate the disease-causing Ryr2 allele and hence rescue catecholaminergic polymorphic ventricular tachycardia.

Objective: To determine if somatic in vivo genome editing using the CRISPR/Cas9 system delivered by adeno-associated viral (AAV) vectors could correct catecholaminergic polymorphic ventricular tachycardia arrhythmias in mice heterozygous for RyR2 mutation R176Q (R176Q/+).

Methods and results: Guide RNAs were designed to specifically disrupt the R176Q allele in the R176Q/+ mice using the SaCas9 ( Staphylococcus aureus Cas9) genome editing system. AAV serotype 9 was used to deliver Cas9 and guide RNA to neonatal mice by single subcutaneous injection at postnatal day 10. Strikingly, none of the R176Q/+ mice treated with AAV-CRISPR developed arrhythmias, compared with 71% of R176Q/+ mice receiving control AAV serotype 9. Total Ryr2 mRNA and protein levels were significantly reduced in R176Q/+ mice, but not in wild-type littermates. Targeted deep sequencing confirmed successful and highly specific editing of the disease-causing R176Q allele. No detectable off-target mutagenesis was observed in the wild-type Ryr2 allele or the predicted putative off-target site, confirming high specificity for SaCas9 in vivo. In addition, confocal imaging revealed that gene editing normalized the enhanced Ca2+ spark frequency observed in untreated R176Q/+ mice without affecting systolic Ca2+ transients.

Conclusions: AAV serotype 9-based delivery of the SaCas9 system can efficiently disrupt a disease-causing allele in cardiomyocytes in vivo. This work highlights the potential of somatic genome editing approaches for the treatment of lethal autosomal-dominant inherited cardiac disorders, such as catecholaminergic polymorphic ventricular tachycardia.

Keywords: allele; electrophysiology; gene editing; mice; mutation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / genetics
  • Animals
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Cas Systems*
  • Calcium Signaling / genetics
  • Dependovirus / genetics
  • Disease Models, Animal
  • Gene Editing / methods*
  • Genetic Predisposition to Disease
  • Genetic Therapy / methods*
  • Genetic Vectors
  • Heart Rate / genetics
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Mutation*
  • Phenotype
  • RNA, Guide / genetics
  • Ryanodine Receptor Calcium Release Channel / genetics*
  • Ryanodine Receptor Calcium Release Channel / metabolism
  • Tachycardia, Ventricular / genetics
  • Tachycardia, Ventricular / metabolism
  • Tachycardia, Ventricular / physiopathology
  • Tachycardia, Ventricular / therapy*


  • RNA, Guide
  • Ryanodine Receptor Calcium Release Channel
  • ryanodine receptor 2. mouse
  • CRISPR-Associated Protein 9

Supplementary concepts

  • Polymorphic catecholergic ventricular tachycardia