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PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys

Zhen-Zhen Chen Jian-Ying Wang Yu Kang Qiao-Yan Yang Xue-Ying Gu Da-Long Zhi Li Yan Cheng-Zu Long Bin Shen Yu-Yu Niu

Zhen-Zhen Chen, Jian-Ying Wang, Yu Kang, Qiao-Yan Yang, Xue-Ying Gu, Da-Long Zhi, Li Yan, Cheng-Zu Long, Bin Shen, Yu-Yu Niu. PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys. Zoological Research, 2021, 42(4): 469-477. doi: 10.24272/j.issn.2095-8137.2021.023
Citation: Zhen-Zhen Chen, Jian-Ying Wang, Yu Kang, Qiao-Yan Yang, Xue-Ying Gu, Da-Long Zhi, Li Yan, Cheng-Zu Long, Bin Shen, Yu-Yu Niu. PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys. Zoological Research, 2021, 42(4): 469-477. doi: 10.24272/j.issn.2095-8137.2021.023

使用成对截短的sgRNA/Cas9-D10A得到PINK1基因突变的食蟹猴

doi: 10.24272/j.issn.2095-8137.2021.023

PINK1 gene mutation by pair truncated sgRNA/Cas9-D10A in cynomolgus monkeys

Funds: This research was supported by the National Key Research and Development Program (2016YFA0101401 and 2018YFA0801400) and Major Basic Research Project of Science and Technology of Yunnan (2019FY002 and 202001BC070001)
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  • 摘要: 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。
  • Figure  1.  Paired Cas9-D10A nickases induce efficient genome editing of PINK1 in cynomolgus monkey embryos

    A: Schematic of sgRNAs targeting PINK1 loci. Guide RNA sequences are underlined and highlighted in red. PAM sequences are highlighted in green. B: Cas9-mediated on-target cleavage of PINK1 by T7E1 cleavage assay. PCR products were amplified and subjected to T7E1 digestion. Samples with cleavage bands are marked with an asterisk. M, marker; WT, wild-type. C: Editing profiles of marked samples in (B). Undigested PCR products from (B) were subjected to TA cloning. Single TA clones were selected and analyzed by DNA sequencing. For WT alleles, PAM sequences are highlighted in green and sgRNA sequences are labeled in red. For alleles with indels, deleted bases are replaced with colons and inserted bases are labelled in lower case and highlighted in blue; deletions (-) and insertions (+). D: Developmental rate in Cas9-D10A-injected embryos is comparable to that in ICSI-treated embryos. ICSI, intracytoplasmic sperm injection embryo; PINK1-Mutant, PINK1 sgRNA injected embryo; 2C, 2-cell embryo; 4C, 4-cell embryo; 8C, 8-cell embryo; M, morula; B, blastula.

    Figure  2.  Paired Cas9-D10A nickases enable one-step generation of PINK1 mutant monkeys

    A: Summary of embryos injected, transferred, impregnated, and birthed. B: Representative images of blastocysts developed from zygotes injected with or without Cas9-D10A mRNA and sgRNA. Scale bars: 200 μm. C: Photo of D10A-M1, -M2, and -M3 (left to right) (taken when the monkeys were 3 years old). D: T7E1 cleavage assay of target site containing DNA products amplified from ear or blood tissue of mutant monkeys (D10A-M1, -M2, and -M3). Top panel represents undigested PCR bands and bottom panel represents digested PCR products. E: Ear; B: Blood; M: Marker. E: Editing profiles of mutant monkeys. Regions containing target sites were amplified from mutant fibroblasts and PCR products were subjected to TA cloning. Single TA clones were selected and analyzed by DNA sequencing. For WT allele, PAM sequences are highlighted in green and sgRNA sequences are labeled in red. For alleles with indels, deleted bases are replaced with colons and inserted bases are labelled in lower case and highlighted in blue; deletions (-) and insertions (+). F: RT-qPCR assay on mutant and WT fibroblasts (GAPDH was used for normalization). Compared with WT monkey, all mutant monkeys showed lower PINK1 mRNA expression. ***: P≤0.001; **: P≤0.01; *: P≤0.1. G: Western blotting assay on mutant and WT fibroblasts. H: Relative PINK1 expression levels were calculated using ImageJ 1.8.0 software. Compared with WT monkey, all mutant monkeys exhibited similar PINK1 protein expression. I: Representative images of immunofluorescence staining of mutant and WT fibroblasts. Scale bars: 200 μm. J: Numbers of total cells and PINK1-positive cells were counted using ImageJ 1.8.0 software.

    Figure  3.  PINK1 mutant fibroblast-derived DA neurons did not show any specific PD-associated phenotypes

    A: Reprogramming of fibroblasts into iPSCs by Sendai virus. Images represent typical cell phenotypes observed on days 0 and 16 after virus transduction and iPSC phenotypes at passage 10. Scale bars: 500 μm. B: Immunofluorescence staining of pluripotency markers OCT4, Nanog, SOX2, and TRA-1-81/60 in iPSCs. Scale bars: 250 μm. C: Teratoma differentiation of iPSCs in immunodeficient mice (NOD/SCID). Left, ectoderm; middle, mesoderm; right, endoderm. Scale bars: 500 μm. D: T7E1 cleavage assay of target sites amplified from three iPSC clones reprogrammed from D10A-M3 fibroblasts. Top panel represents undigested PCR bands, and bottom panel represents digested PCR products. E: Genotypes of iPSC-1 and iPSC-2. Two iPSCs showing different lengths of DNA fragments. F: Induction process of iPSCs to DA neurons. During induction, different differentiation and maturation media need to be replaced. After 30 days, DA neurons can be obtained. Scale bars: 500 μm. G: Representative images showing immunofluorescence staining of D10A-M3 and WT iPSC-derived DA neuron markers NeuN, Foxa2, TH, and GIRK2. Scale bars: 250 μm. H: Relative expression levels in (G) were calculated via ImageJ 1.8.0 software. No significant differences were observed between the proportion of mature DA neurons in D10A-M3 and WT during induction. I: Mitochondrial morphology of D10A-M3 and WT-derived DA neurons under electron microscopy. Compared with WT monkey, D10A-M3 exhibited similar mitochondrial morphology. Arrows, mitochondria of D10A-M3 and WT-derived neurons Scale bars: 1 μm.

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出版历程
  • 收稿日期:  2021-05-09
  • 录用日期:  2021-06-28
  • 网络出版日期:  2021-06-30
  • 刊出日期:  2021-07-18

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