Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption

Anne Katrine Johansen, Bas Molenaar, Danielle Versteeg, Ana Rita Leitoguinho, Charlotte Demkes, Bastiaan Spanjaard, Hesther de Ruiter, Farhad Akbari Moqadam, Lieneke Kooijman, Lorena Zentilin, Mauro Giacca, Eva van Rooij

Research output: Contribution to journal/periodicalArticleScientificpeer-review

50 Citations (Scopus)


RATIONALE: CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9)-based DNA editing has rapidly evolved as an attractive tool to modify the genome. Although CRISPR/Cas9 has been extensively used to manipulate the germline in zygotes, its application in postnatal gene editing remains incompletely characterized.

OBJECTIVE: To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice.

METHODS AND RESULTS: We generated cardiomyocyte-specific Cas9 mice and demonstrated that Cas9 expression does not affect cardiac function or gene expression. As a proof-of-concept, we delivered short guide RNAs targeting 3 genes critical for cardiac physiology, Myh6, Sav1, and Tbx20, using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of short guide RNA exposure or the level of Cas9 expression. DNA sequencing analysis revealed target-dependent mutations that were highly reproducible across mice resulting in differential rates of in- and out-of-frame mutations. Finally, we applied a dual short guide RNA approach to effectively delete an important coding region of Sav1, which increased the editing efficiency.

CONCLUSIONS: Our results indicate that the effect of postnatal CRISPR/Cas9-based cardiac gene editing using adeno-associated virus serotype 9 to deliver a single short guide RNA is target dependent. We demonstrate a mosaic pattern of gene disruption, which hinders the application of the technology to study gene function. Further studies are required to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.

Original languageEnglish
Pages (from-to)1168-1181
Number of pages14
JournalCirculation Research
Issue number10
Publication statusPublished - 27 Oct 2017


  • Animals
  • Animals, Newborn
  • Base Sequence
  • CRISPR-Cas Systems
  • Cells, Cultured
  • Dependovirus
  • Gene Editing
  • Gene Transfer Techniques
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Mice, Transgenic
  • Myocytes, Cardiac
  • NIH 3T3 Cells
  • RNA, Guide
  • Journal Article


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