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
AUTHORS (12)
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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