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

TY - JOUR

T1 - Postnatal Cardiac Gene Editing Using CRISPR/Cas9 With AAV9-Mediated Delivery of Short Guide RNAs Results in Mosaic Gene Disruption

AU - Johansen, Anne Katrine

AU - Molenaar, Bas

AU - Versteeg, Danielle

AU - Leitoguinho, Ana Rita

AU - Demkes, Charlotte

AU - Spanjaard, Bastiaan

AU - de Ruiter, Hesther

AU - Akbari Moqadam, Farhad

AU - Kooijman, Lieneke

AU - Zentilin, Lorena

AU - Giacca, Mauro

AU - van Rooij, Eva

N1 - © 2017 American Heart Association, Inc.

PY - 2017/10/27

Y1 - 2017/10/27

N2 - 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.

AB - 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.

KW - Animals

KW - Animals, Newborn

KW - Base Sequence

KW - CRISPR-Cas Systems

KW - Cells, Cultured

KW - Dependovirus

KW - Gene Editing

KW - Gene Transfer Techniques

KW - Mice

KW - Mice, Inbred C57BL

KW - Mice, Knockout

KW - Mice, Transgenic

KW - Myocytes, Cardiac

KW - NIH 3T3 Cells

KW - RNA, Guide

KW - Journal Article

U2 - 10.1161/CIRCRESAHA.116.310370

DO - 10.1161/CIRCRESAHA.116.310370

M3 - Article

C2 - 28851809

SN - 0009-7330

VL - 121

SP - 1168

EP - 1181

JO - Circulation Research

JF - Circulation Research

IS - 10

ER -