PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys
PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys作者机构：Faculty of Life Science and TechnologyKunming University of Science and TechnologyKunmingYunnan 650500China State Key Laboratory of Primate Biomedical ResearchInstitute of Primate Translational MedicineKunming University of Science and TechnologyKunmingYunnan 650500China State Key Laboratory of Reproductive MedicineDepartment of Prenatal DiagnosisWomen’s Hospital of Nanjing Medical UniversityNanjing Maternity and Child Health HospitalNanjing Medical UniversityNanjingJiangsu 211166China Leon H Charney Division of CardiologyNew York University School of MedicineNew YorkNY 10016USA Department of DermatologyXijing HospitalFourth Military Medicine UniversityXi’anShaanxi 710032China
出 版 物：《Zoological Research》 (动物学研究（英文）)
年 卷 期：2021年第42卷第4期
页 面：469-477页
核心收录：
学科分类：0710[理学-生物学] 0830[工学-环境科学与工程（可授工学、理学、农学学位）] 1002[医学-临床医学] 0905[农学-畜牧学] 0906[农学-兽医学] 100204[医学-神经病学] 0833[工学-城乡规划学] 10[医学] 0713[理学-生态学] 0834[工学-风景园林学（可授工学、农学学位）]
基 金：supported by the National Key Research and Development Program(2016YFA0101401 and 2018YFA0801400) Major Basic Research Project of Science and Technology of Yunnan(2019FY002 and 202001BC070001)。
主 题：Cas9-D10A Cynomolgus PINK1
摘 要：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.
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