<i>PINK1</i> 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

doi:10.24272/j.issn.2095-8137.2021.023

Volume 42 Issue 4

Jul. 2021

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Article Navigation > Zoological Research > 2021 > 42(4): 469-477

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

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

doi: 10.24272/j.issn.2095-8137.2021.023

Zhen-Zhen Chen^{1, 2},
Jian-Ying Wang³,
Yu Kang^{1, 2},
Qiao-Yan Yang⁴,
Xue-Ying Gu³,
Da-Long Zhi^{2, 5},
Li Yan²,
Cheng-Zu Long⁴,
Bin Shen^{3
,
,},
Yu-Yu Niu^{1, 2
,
,}

1.
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
2.
State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China
3.
State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Hospital, Nanjing Medical University, Nanjing, Jiangsu 211166, China
4.
Leon H Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA
5.
Department of Dermatology, Xijing Hospital, Fourth Military Medicine University, Xi’an, Shaanxi 710032, China

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)

More Information

Corresponding author: E-mail: binshen@njmu.edu.cn; niuyy@lpbr.cn
Received Date: 2021-05-09
Accepted Date: 2021-06-28

Published Online: 2021-06-30

Publish Date: 2021-07-18

Abstract

Abstract

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.
- Cas9-D10A,
- Cynomolgus,
- PINK1

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References(32)

References

[1]	Brogna S, Wen JK. 2009. Nonsense-mediated mRNA decay (NMD) mechanisms. Nature Structural & Molecular Biology, 16(2): 107−113.
[2]	Chan AWS. 2013. Progress and prospects for genetic modification of nonhuman primate models in biomedical research. ILAR Journal, 54(2): 211−223. doi: 10.1093/ilar/ilt035
[3]	Dawson TM, Ko HS, Dawson VL. 2010. Genetic animal models of Parkinson's disease. Neuron, 66(5): 646−661. doi: 10.1016/j.neuron.2010.04.034
[4]	Dianov GL, Hübscher U. 2013. Mammalian base excision repair: the forgotten archangel. Nucleic Acids Research, 41(6): 3483−3490. doi: 10.1093/nar/gkt076
[5]	Fu YF, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nature Biotechnology, 32(3): 279−284. doi: 10.1038/nbt.2808
[6]	Goldstein DS, Sharabi Y. 2019. The heart of PD: lewy body diseases as neurocardiologic disorders. Brain Research, 1702: 74−84. doi: 10.1016/j.brainres.2017.09.033
[7]	Gopalappa R, Suresh B, Ramakrishna S, Kim H. 2018. Paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. Nucleic Acids Research, 46(12): e71. doi: 10.1093/nar/gky222
[8]	Guilinger JP, Thompson DB, Liu DR. 2014. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature Biotechnology, 32(6): 577−582. doi: 10.1038/nbt.2909
[9]	Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V et al. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology. 31(9):827-32.
[10]	Joanna Z, Magdalena H, Agnieszka NT, Jacek J, Ryszard S, Zdzisław S, et al. 2018. The production of UL16-binding protein 1 targeted pigs using CRISPR technology. 3 Biotech, 8(1): 70. doi: 10.1007/s13205-018-1107-4
[11]	Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng ZL, et al. 2016. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature, 529(7587): 490−495. doi: 10.1038/nature16526
[12]	Lasbleiz C, Mestre-Francés N, Devau G, Luquin MR, Tenenbaum L, Kremer EJ, et al. 2019. Combining gene transfer and nonhuman primates to better understand and treat parkinson's disease. Frontiers in Molecular Neuroscience, 12: 10. doi: 10.3389/fnmol.2019.00010
[13]	Lee JH, Kim SW, Park TS. 2017. Myostatin gene knockout mediated by Cas9-D10A nickase in chicken DF1 cells without off-target effect. Asian-Australasian Journal of Animal Sciences, 30(5): 743−748.
[14]	Morais VA, Haddad D, Craessaerts K, De Bock PJ, Swerts J, Vilain S, et al. 2014. PINK1 loss-of-function mutations affect mitochondrial complex I activity via NdufA10 ubiquinone uncoupling. Science, 344(6180): 203−207. doi: 10.1126/science.1249161
[15]	Nakamura K, Edwards RH. 2007. Physiology versus pathology in Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America, 104(29): 11867−11868. doi: 10.1073/pnas.0704254104
[16]	Niu YY, Shen B, Cui YQ, Chen YC, Wang JY, Wang L, et al. 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156(4): 836−843. doi: 10.1016/j.cell.2014.01.027
[17]	Okita K, Ichisaka T, Yamanaka S. 2007. Generation of germline-competent induced pluripotent stem cells. Nature, 448(7151): 313−317. doi: 10.1038/nature05934
[18]	Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR. 2013. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology. 31(9):839-43.
[19]	Pickrell AM, Youle RJ. 2015. The roles of PINK1, parkin, and mitochondrial fidelity in Parkinson's disease. Neuron, 85(2): 257−273. doi: 10.1016/j.neuron.2014.12.007
[20]	Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, et al. 2017. Parkinson disease. Nature Reviews Disease Primers, 3(1): 17013. doi: 10.1038/nrdp.2017.13
[21]	Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. 2013. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154(6): 1380−1389. doi: 10.1016/j.cell.2013.08.021
[22]	Reed X, Bandrés-Ciga S, Blauwendraat C, Cookson MR. 2019. The role of monogenic genes in idiopathic Parkinson's disease. Neurobiology of Disease, 124: 230−239. doi: 10.1016/j.nbd.2018.11.012
[23]	Schmid-Burgk JL, Gao LY, Li D, Gardner Z, Strecker J, Lash B, et al. 2020. Highly Parallel Profiling of Cas9 Variant Specificity. Molecular Cell, 78(4): 794−800.e8. doi: 10.1016/j.molcel.2020.02.023
[24]	Shen B, Zhang WS, Zhang J, Zhou JK, Wang JY, Chen L, et al. 2014. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nature Methods, 11(4): 399−402. doi: 10.1038/nmeth.2857
[25]	Song X, Huang H, Xiong ZQ, Ai LZ, Yang S. 2017. CRISPR-Cas9^D10A nickase-assisted genome editing in Lactobacillus casei. Applied and Environmental Microbiology, 83(22): e01259-17. doi: 10.1128/aem.01259-17
[26]	Takahashi K, Yamanaka S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4): 663−676. doi: 10.1016/j.cell.2006.07.024
[27]	Unoki M, Nakamura Y. 2001. Growth-suppressive effects of BPOZ and EGR2, two genes involved in the PTEN signaling pathway. Oncogene, 20(33): 4457−4465. doi: 10.1038/sj.onc.1204608
[28]	Vermilyea SC, Emborg ME. 2018. The role of nonhuman primate models in the development of cell-based therapies for Parkinson's disease. Journal of Neural Transmission, 125(3): 365−384. doi: 10.1007/s00702-017-1708-9
[29]	Yang WL, Liu YB, Tu ZC, Xiao C, Yan S, Ma XS, et al. 2019. CRISPR/Cas9-mediated PINK1 deletion leads to neurodegeneration in rhesus monkeys. Cell Research, 29(4): 334−336. doi: 10.1038/s41422-019-0142-y
[30]	Youle RJ, Van Der Bliek AM. 2012. Mitochondrial fission, fusion, and stress. Science, 337(6098): 1062−1065. doi: 10.1126/science.1219855
[31]	Zhang XH, Tee LY, Wang XG, Huang QS, Yang SH. 2015. Off-target effects in CRISPR/Cas9-mediated genome engineering. Molecular Therapy-Nucleic Acids, 4: e264. doi: 10.1038/mtna.2015.37
[32]	Zhou XQ, Xin JG, Fan NN, Zou QJ, Huang J, Ouyang Z, et al. 2015. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences, 72(6): 1175−1184. doi: 10.1007/s00018-014-1744-7