<i>PINK1</i> gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys
Zhenzhen Chen, 昆明理工大学生命科学与技术学院，云南 昆明 650500，中国, Jian-Ying Wang, Yu Kang, Qiaoyan Yang, Xueying Gu, Dalong Zhi, Yan Li, Chengzu Long, Bin Shen, Yuyu Niu, 昆明理工大学灵长类转化医学研究院非人灵长类生物医学国家重点实验室，云南 昆明 650500，中国, 南京医科大学附属南京妇幼保健院产前诊断科，生殖医学国家重点实验室，江苏 南京 211166，中国, 第四军医大学西京医院皮肤科，陕西 西安 710032，中国
Abstract
Mutations of <i>PTEN-induced kinase I</i> (<i>PINK1</i>) cause early-onset Parkinson’s disease (PD) with selective neurodegeneration in humans. However, current <i>PINK1</i> knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients. This suggests that generating <i>PINK1</i> 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 <i>PINK1</i> gene. We achieved precise and efficient gene editing of the target site in three newborn cynomolgus monkeys. The frame shift mutations of <i>PINK1</i> 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 <i>PINK1</i> gene exon 2 did not impact protein expression.