Mutations of PINK1 cause early-onset Parkinson's disease (PD) with selective neurodegeneration in humans. However, current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients, indicating that it is essential to generate PINK1 disease models in non-human primates (NHPs) that are highly close to humans, to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both enabling the reduction of the off-target effect without apparently compromising the on-target editing, are two optimized strategies in applying the CRISPR/Cas9 system to establish disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target PINK1 gene. We show precise and efficient gene editing of the target site in the three new born cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts leaded to the reduction of the mRNA, However, western-blot and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in the wild-type fibroblasts. We further reprogramed mutant fibroblast into induced pluripotent stem cells (iPSCs) and they show similar ability of differentiation into dopamine (DA) neurons. Taken together, our results showed that coinjection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enable the generation of human disease models in NHPs and the target editing by paired truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact the protein expression.

中文翻译：

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食蟹猴中配对的截短的sgRNA / Cas9-D10A突变PINK1基因

PINK1突变会导致人类早期帕金森氏病（PD），并伴有选择性神经变性。然而，目前的PINK1基因敲除小鼠和猪模型无法概括在PD患者中观察到的典型神经变性表型，这表明在高度接近人类的非人类灵长类动物（NHP）中生成PINK1疾病模型至关重要，以研究PINK1在灵长类动物大脑中的独特功能。配对的单向导RNA（sgRNA）/ Cas9-D10A切口酶和截短的sgRNA / Cas9都可以降低脱靶效应，而不会明显影响靶标编辑，这是应用CRISPR / Cas9系统建立双链的两个优化策略疾病动物模型。这里，我们结合了这两种策略，并将Cas9-D10A mRNA和两个截短的sgRNA注射入单细胞阶段的食蟹猴合子以靶向PINK1基因。我们展示了三只新生食蟹猴中目标部位的精确和有效的基因编辑。突变成纤维细胞中PINK1的移码突变导致mRNA的降低，但是，western印迹和免疫荧光染色证实PINK1蛋白水平与野生型成纤维细胞相当。我们进一步将突变的成纤维细胞重编程为诱导性多能干细胞（iPSCs），它们显示出类似的分化为多巴胺（DA）神经元的能力。在一起