Mutations of PTEN-induced kinase I (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. This suggests that generating PINK1 disease models in non-human primates (NHPs) that are close to humans is essential to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both of which can reduce off-target effects without compromising on-target editing, are two optimized strategies in the CRISPR/Cas9 system for establishing 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 the PINK1 gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts led to a reduction in mRNA. However, western blotting and immunofluorescence staining confirmed the PINK1 protein levels were comparable to that in wild-type fibroblasts. We further reprogramed mutant fibroblasts into induced pluripotent stem cells (iPSCs), which showed similar ability to differentiate into dopamine (DA) neurons. Taken together, our results showed that co-injection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enabled the generation of human disease models in NHPs and target editing by pair truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact protein expression.
PINK1 (PTEN-induced putative kinase1)蛋白的突变导致可能会导致早发性帕金森病 (Parkinson's disease，PD) 和选择性神经变性。然而，目前的PINK1基因敲除小鼠和猪模型无法重现在PD患者中观察到的典型神经退行性表型。这表明在接近人类的非人类灵长类动物 (non human primates, NHPs) 中生成 PINK1 疾病模型对于研究 PINK1在灵长类动物大脑中的独特功能至关重要。使用Cas9-D10A核酸内切酶和成对的sgRNA (single guide RNA)都可以在不影响目标编辑的情况下减少脱靶效应，是CRISPR/Cas9系统中用于建立疾病动物模型的两种优化策略。在这里，我们将两种策略结合起来，将Cas9-D10A 的mRNA和两个截短的sgRNA注射到单细胞阶段食蟹猴受精卵中，以靶向编辑PINK1基因。我们在三只新生食蟹猴中实现了对目标位点的精确高效的基因编辑，在突变的成纤维细胞中，PINK1基因的移码突变导致其mRNA的表达量减少。然而，蛋白质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新编程为诱导多能干细胞 (induced pluripotent stem cell，iPSC)，两者显示出相似的分化为多巴胺 (dopaminergic, DA) 神经元的能力。综上所述，我们的结果表明，将Cas9-D10A切口酶mRNA和sgRNA共同注射到单细胞阶段食蟹猴胚胎中，能够使用NHPs构建人类疾病模型，并通过PINK1基因外显子中截短的sgRNA/Cas9-D10A对进行2号外显子的靶向编辑并不影响PINK1蛋白质表达。.
Keywords: Cas9-D10A; Cynomolgus; PINK1.