From the Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (A.K.J., B.M., D.V., A.R.L., C.D., B.S., H.d.R., F.A.M., L.K., E.v.R.) and Department of Cardiology (D.V., C.D., E.v.R.), University Medical Center Utrecht, The Netherlands; and International Centre for Genetic Engineering and Biotechnology, Trieste, Italy (L.Z., M.G.).
Circ Res. 2017 Oct 27;121(10):1168-1181. doi: 10.1161/CIRCRESAHA.116.310370. Epub 2017 Aug 29.
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.
To evaluate the feasibility of CRISPR/Cas9-based cardiac genome editing in vivo in postnatal mice.
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, , , and , using a cardiotropic adeno-associated viral vector 9. Despite a similar degree of DNA disruption and subsequent mRNA downregulation, only disruption of 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 , which increased the editing efficiency.
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.
基于 CRISPR/Cas9（规律成簇间隔短回文重复/CRISPR 相关蛋白 9）的 DNA 编辑技术迅速发展，成为一种有吸引力的基因编辑工具。虽然 CRISPR/Cas9 已被广泛用于操纵受精卵中的种系细胞，但它在产后基因编辑中的应用尚未完全确定。
评估 CRISPR/Cas9 介导的产后小鼠体内心脏基因组编辑的可行性。
我们构建了心肌细胞特异性 Cas9 小鼠，并证实 Cas9 表达不会影响心脏功能或基因表达。作为概念验证，我们使用心肌靶向的腺相关病毒载体 9 传递靶向 3 个对心脏生理至关重要的基因（ 、 、 和 ）的短向导 RNA。尽管 DNA 破坏程度和随后的 mRNA 下调程度相似，但只有 的破坏足以诱导心脏表型，而与短向导 RNA 暴露或 Cas9 表达水平无关。DNA 测序分析显示，靶基因依赖性突变在小鼠中具有高度可重复性，导致错义突变和无义突变的比率不同。最后，我们应用双重短向导 RNA 方法有效删除了 中一个重要的编码区域，从而提高了编辑效率。
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 的产后 CRISPR/Cas9 心脏基因编辑的效果取决于靶基因。我们证明了基因破坏的镶嵌模式，这限制了该技术在研究基因功能中的应用。需要进一步研究来扩展 CRISPR/Cas9 的多功能性，使其成为一种在体内研究新的心脏基因功能的强大工具。
