Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice.
Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice.
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DOI：
10.3791/57560
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发表时间：
2018-08
期刊：
影响因子：
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通讯作者：
Li Xu;Yandi Gao;Y. Lau;Renzhi Han
中科院分区：
文献类型：
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作者：
Li Xu;Yandi Gao;Y. Lau;Renzhi Han
聚集的、有规则间隔的、短的、回文重复（CRISPR）系统极大地促进了来自各种物种的培养细胞和活生物体的基因组工程。CRISPR技术也被探索作为许多人类疾病的新疗法。最近的研究证明了基于基因编辑的治疗方法在小鼠模型上治疗杜氏肌营养不良症（DMD）的可行性和有效性，这表明概念验证数据非常令人鼓舞。特别是，静脉和腹腔注射重组腺相关病毒（rAAV）血清型rh.74（rAAVrh.74）能够有效地通过心脏递送金黄色葡萄球菌crispr相关蛋白9 （SaCas9）和两个引导rna （gRNA）来删除小鼠Dmd基因23外显子中具有突变密码子的基因组区域。当gRNA被设计为靶向基因编码区域时，同样的方法也可以用于敲除感兴趣的基因，并在出生后的小鼠中研究它们的心脏功能。在本协议中，我们详细展示了如何设计rAAVrh。74-CRISPR载体及如何在新生小鼠中实现高效的心脏传递。
The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.