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

• DOI: 10.1161/circresaha.116.310370
• Publication Date: 2017-08-30T00:50:16Z
• Authors: Anne Katrine Joha...; Bas Molenaar; Danielle Versteeg; Ana Rita Leitoguinho; Charlotte Demkes; Bastiaan Spanjaard; Hesther de Ruiter; Farhad Akbari Moq...; Lieneke Kooijman; Lorena Zentilin; Mauro Giacca; Eva van Rooij
• Keywords: 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

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.