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

• Published in: Circulation research 2017-10, Vol.121 (10), p.1168-1181
• Main Authors: Johansen, Anne Katrine, Molenaar, Bas, Versteeg, Danielle, Leitoguinho, Ana Rita, Demkes, Charlotte, Spanjaard, Bastiaan, de Ruiter, Hesther, Moqadam, Farhad Akbari, Kooijman, Lieneke, Zentilin, Lorena, Giacca, Mauro, van Rooij, Eva
• Format: Article
• Language: English
• Subjects: Animals; Animals, Newborn; Base Sequence; Cardiomyocytes; Cells, Cultured; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; Dependovirus - genetics; DNA; DNA sequencing; Gene disruption; Gene Editing - methods; Gene expression; Gene Transfer Techniques; Genome editing; Genomes; Heart; Heart diseases; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mutation; Myocytes, Cardiac - physiology; NIH 3T3 Cells; Phenotypes; RNA, Guide, CRISPR-Cas Systems - administration & dosage; RNA, Guide, CRISPR-Cas Systems - genetics; Zygotes
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.116.310370

RATIONALE:CRISPR/Cas9-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 (sgRNAs) targeting three genes critical for cardiac physiology, Myh6, Sav1 and Tbx20, using a cardiotropic adeno-associated viral vector (AAV9). Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of sgRNA 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 sgRNA approach to effectively delete en 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 AAV9 to deliver a single sgRNA 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 in order to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.