Rationale: CRISPR/Cas9-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 (sgRNAs) targeting three genes critical for cardiac physiology, Myh6, Sav1 and Tbx20, using a cardiotropic adeno-associated viral vector (AAV9). Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of Myh6 was sufficient to induce a cardiac phenotype, irrespective of sgRNA 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 sgRNA approach to effectively delete en 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 AAV9 to deliver a single sgRNA 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 in order to expand the versatility of CRISPR/Cas9 as a robust tool to study novel cardiac gene functions in vivo.

基本原理：基于CRISPR / Cas9的DNA编辑已迅速发展成为修饰基因组的有吸引力的工具。尽管CRISPR / Cas9已被广泛用于操纵受精卵中的种系，但其在产后基因编辑中的应用仍不完整。目的：评估基于CRISPR / Cas9的心脏基因组在出生后小鼠体内编辑的可行性。方法和结果：我们产生了心肌细胞特异性Cas9小鼠，并证明Cas9表达不影响心脏功能或基因表达。作为概念的证明，我们提供了针对三种对心脏生理至关重要的基因Myh6，Sav1和Tbx20的短指导RNA（sgRNA），使用与心脏有关的腺相关病毒载体（AAV9）。尽管存在相似程度的DNA破坏和随后的mRNA下调，但无论是sgRNA暴露还是Cas9表达水平，只有Myh6破坏才足以诱导心脏表型。DNA测序分析显示，靶标依赖性突变在小鼠之间具有很高的重现性，导致框内和框外突变的发生率不同。最后，我们应用双重sgRNA方法有效删除了Sav1的重要编码区，从而提高了编辑效率。结论：我们的结果表明，使用AAV9传递单个sgRNA的基于出生后CRISPR / Cas9的心脏基因编辑的效果取决于靶标。我们展示了基因破坏的马赛克模式，这阻碍了该技术在研究基因功能中的应用。为了扩大CRISPR / Cas9的多功能性，作为在体内研究新型心脏基因功能的有力工具，需要进一步的研究。