基于多重CRISPR/Cas9的基因组编辑用于纠正导致杜氏肌营养不良症的肌营养不良蛋白突变。
Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy.
作者信息
Ousterout David G, Kabadi Ami M, Thakore Pratiksha I, Majoros William H, Reddy Timothy E, Gersbach Charles A
机构信息
Department of Biomedical Engineering, Duke University, Room 136 Hudson Hall, Box 90281, Durham, North Carolina 27708, USA.
Program in Computational Biology and Bioinformatics, Duke University, Durham, North Carolina 27708, USA.
出版信息
Nat Commun. 2015 Feb 18;6:6244. doi: 10.1038/ncomms7244.
The CRISPR/Cas9 genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here, we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45-55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene-editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations.
CRISPR/Cas9基因组编辑平台是一种用于纠正遗传性疾病遗传基础的很有前景的技术。CRISPR/Cas9系统的多功能性、高效性和多重编辑能力使得各种原本具有挑战性的基因校正策略成为可能。在此，我们使用CRISPR/Cas9系统在携带导致杜氏肌营养不良症（DMD）的肌营养不良蛋白突变的细胞中恢复肌营养不良蛋白基因的表达。我们设计单链或多重引导RNA（sgRNAs），通过靶向45-55外显子的突变热点并在外显子内引入移码或删除一个或多个外显子，来恢复肌营养不良蛋白的阅读框。在对DMD患者成肌细胞进行基因编辑后，肌营养不良蛋白的表达在体外得以恢复。将经过基因校正的患者细胞移植到免疫缺陷小鼠体内后，在体内也检测到了人肌营养不良蛋白。重要的是，CRISPR/Cas9系统独特的多重基因编辑能力有助于产生一个单一的大缺失，该缺失可校正高达62%的DMD突变。
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