Cruz Lilian, György Bence, Cheah Pike See, Kleinstiver Benjamin P, Eimer William A, Garcia Sara P, Sharma Nutan, Ozelius Laurie J, Bragg D Cristopher, Joung J Keith, Norberto de Souza Osmar, Macedo Timmers Luis Fernando Saraiva, Breakefield Xandra O
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Neurobiology Department, Harvard Medical School, Boston, MA, USA; Institute of Molecular and Clinical Ophthalmology Basel, Basel, Switzerland; Department of Ophthalmology, University of Basel, Basel, Switzerland.
Mol Ther Nucleic Acids. 2020 Sep 4;21:1-12. doi: 10.1016/j.omtn.2020.05.009. Epub 2020 May 15.
Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
大多数患有DYT1肌张力障碍的个体在TOR1A基因中存在杂合性3碱基对缺失（c.907_909delGAG）。该突变似乎通过一种显性负性机制发挥作用，损害正常的torsinA功能，并且有人提出减少突变型torsinA可能会使torsinA活性恢复正常。在本研究中，我们使用来自化脓性链球菌的工程化Cas9变体（SpCas9-VRQR）靶向TOR1A基因中的突变，以破坏DYT1患者成纤维细胞中的突变型torsinA。对于大多数常见的非同源末端连接（NHEJ）编辑，DYT1等位基因的选择性靶向效率很高，导致预测的提前终止密码子，torsinA C末端缺失（缺失302-332个氨基酸）。结构分析预测，由于与ATP酶活性和与LULL1结合相关的残基缺失，这种截短的torsinA处于功能失活状态。免疫印迹显示，在经Cas9编辑的DYT1成纤维细胞中torsinA蛋白水平降低，使用单纯疱疹病毒感染的功能测定表明表型恢复到对照成纤维细胞中观察到的状态。这些发现表明，使用CRISPR-Cas9选择性破坏突变型TOR1A等位基因可使突变型torsinA失活，使剩余的野生型torsinA发挥正常功能。
