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.

中文翻译：

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使用CRISPR / Cas9和AAV9介导的短链RNA传递的产后心脏基因编辑导致镶嵌基因破坏的新颖性和意义

原理：基于CRISPR / Cas9（聚簇的规则间隔的回文重复序列/ CRISPR相关蛋白9）的DNA编辑已迅速发展成为一种有吸引力的修饰基因组的工具。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。作为概念验证，我们使用了一种与心脏有关的腺相关病毒载体9，提供了针对3个对心脏生理至关重要的基因Myh6， Sav1和Tbx20的短指导RNA，尽管DNA破坏的程度和随后的mRNA下调相似，仅破坏Myh6不论短链RNA暴露或Cas9表达水平如何，均足以诱导心脏表型。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，我们应用双重短导RNA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，使用腺相关病毒血清型9传递单个短向导RNA的基于CRISPR / Cas9的产后基于心脏基因编辑的效果是靶标依赖性的。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。需要进一步的研究来扩展CRISPR / Cas9的多功能性，将其作为研究体内新的心脏基因功能的有力工具。