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

0301 basic medicine sequence analysis, DNA Physiology Cells Knockout Mice, Transgenic RNA, Guide, CRISPR-Cas Systems Inbred C57BL Transgenic Mice 03 medical and health sciences Journal Article molecular biology RNA, Guide Animals CRISPR-Cas System Myocytes, Cardiac myocytes, cardiac NIH 3T3 Cell Cells, Cultured Gene Editing Mice, Knockout Myocytes 0303 health sciences Cultured Base Sequence gene editing Animal Gene Transfer Techniques Gene Transfer Technique Dependovirus Newborn Dependoviru Mice, Inbred C57BL Animals, Newborn clustered regularly interspaced short palindromic repeats; gene editing; molecular biology; myocytes, cardiac; sequence analysis, DNA; Animals; Animals, Newborn; Base Sequence; CRISPR-Cas Systems; Cells, Cultured; Dependovirus; Gene Editing; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Myocytes, Cardiac; NIH 3T3 Cells; RNA, Guide; Gene Transfer Techniques; Physiology; Cardiology and Cardiovascular Medicine NIH 3T3 Cells RNA CRISPR-Cas Systems Cardiology and Cardiovascular Medicine Cardiac Guide clustered regularly interspaced short palindromic repeat
DOI: 10.1161/circresaha.116.310370 Publication Date: 2017-08-30T00:50:16Z
ABSTRACT
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.
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