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 - Moqadam, Farhad Akbari
AU - Kooijman, Lieneke
AU - Zentilin, Lorena
AU - Giacca, Mauro
AU - Van Rooij, Eva
PY - 2017/1/1
Y1 - 2017/1/1
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 - Cardiac
KW - Clustered regularly interspaced short palindromic repeats
KW - DNA
KW - Gene editing
KW - Molecular biology
KW - Myocytes
KW - Sequence analysis
UR - http://www.scopus.com/inward/record.url?scp=85032645253&partnerID=8YFLogxK
U2 - 10.1161/CIRCRESAHA.116.310370
DO - 10.1161/CIRCRESAHA.116.310370
M3 - Article
C2 - 28851809
AN - SCOPUS:85032645253
SN - 0009-7330
VL - 121
SP - 1168
EP - 1181
JO - Circulation Research
JF - Circulation Research
IS - 10
ER -