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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (6): 31-39    DOI: 10.13523/j.cb.2001063
技术与方法     
小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立 *
周勤1,2,王爽1,2,张婷1,2,李善刚1,2,陈永昌1,2,***()
1 昆明理工大学生命科学与技术学院 灵长类转化医学研究院 昆明 650500
2 云南中科灵长类生物医学重点实验室 昆明 650500
T158M Single Base Editing of MECP2 Gene in Murine and Rhesus Monekey’s Embryos
ZHOU Qin1,2,WANG Shuang1,2,ZHANG Ting1,2,LI Shan-gang1,2,CHEN Yong-chang1,2,***()
1 College of Life Science and Technology, Institute of Primate Translational,Kunming University of Science and Technology,Kunming 650500,China
2 Yunnan Key Laboratory of Primate Biomedical Research,Kunming 650500,China
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摘要：

目的 利用单碱基编辑技术定点修饰MECP2基因T158M位点,获得单一定点突变类型的啮齿类及非人灵长类胚胎,为建立模拟临床上Rett综合征的疾病动物模型奠定基础。方法 利用单碱基编辑技术构建3对T158M-sgRNA质粒,并分别与BE系统(BE3或BE4max)质粒共转染293T细胞筛选高效的sgRNA,通过显微注射将体外转录的mRNA以不同的浓度组合注射到ICR小鼠和猕猴受精卵中,检测小鼠和猕猴胚胎发育率和编辑效率,评估BE3和BE4max的工作效率及最优浓度组合。结果 小鼠胚胎上BE4max(ng/μl)∶sgRNA(ng/μl) = 100∶50浓度组合时,小鼠胚胎发育率和编辑效率最佳,同时该浓度组合在猕猴胚胎上也获得了有效编辑效率。结论 成功在小鼠及猕猴胚胎上实现了MECP2基因T158M位点上C→T的转变,为后期建立T158M突变的RTT动物模型建立了稳定的胚胎编辑体系。
关键词： Rett综合征;  单碱基编辑;  非人灵长类;  编辑效率    
Abstract:

Objective: The T158M site of MECP2 gene was modified by using single-base editing technique to obtain single site-directed mutant rodent and non-human primate embryos,which lays a foundation for the establishment of an animal disease model simulating Rett syndrome in clinical practice.Methods: Three pairs of T158M-sgRNA plasmids were constructed using single-base editing technique,and co-transfected 293T cells with BE system(BE3 or BE4max)plasmids to screen highly-efficient sgRNAs.In vitro transcribed mRNAs were injected at different concentrations by microinjection to ICR mice and macaque fertilized eggs to measure the embryo development rate and editing efficiency of mouse and macaque and to evaluate the working efficiency of BE3 and BE4max and optimal concentration combination.Results: The mouse embryo development rate and editing efficiency are best when the concentration ratio of BE4max(ng/μl)∶sgRNA(ng/μl)on mouse embryos is 100∶50,and this concentration combination also shows effective editing efficiency on rhesus monkey embryo.Conclusion: The transition of C→T at position T158M of the MECP2 gene were successfully achieved in mouse and macaques embryos,establishing a stable embryo editing system for the later establishment of a T158M mutant RTT animal model.
Key words: Rett syndrome    Single base editing    Non-human primate    Editing efficiency
收稿日期: 2020-01-26 出版日期: 2020-06-23
ZTFLH:  Q291  
基金资助: * 国家自然科学基金(U1602224)
通讯作者: 陈永昌     E-mail: chenyc@lpbr.cn
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引用本文:

周勤,王爽,张婷,李善刚,陈永昌. 小鼠及猕猴胚胎MECP2基因T158M单碱基突变体系的建立 *[J]. 中国生物工程杂志, 2020, 40(6): 31-39.

ZHOU Qin,WANG Shuang,ZHANG Ting,LI Shan-gang,CHEN Yong-chang. T158M Single Base Editing of MECP2 Gene in Murine and Rhesus Monekey’s Embryos. China Biotechnology, 2020, 40(6): 31-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001063    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I6/31

Target ID Primer sequences (5'-3')
Target 1 Forward:ACCGGACTTCACGGTAACTGGGAGAGGG
Reward:AAACCCCTCTCCCAGTTACCGTGAAGTC
Target 2 Forward:ACCGGACTTCACGGTAACTGGGAGAGG
Reward:AAACCCTCTCCCAGTTACCGTGAAGTC
Target 3 Forward:ACCGGACCGTGAAGTCAAAATCATTAGG
Reward:AAACCCTAATGATTTTGACTTCACGGTC
表1  针对158位点设计的Oligo DNA
引物信息 引物序列(5'-3')
Homo-MECP2-F CATCCGCTCTGCCCTATCTC
Homo-MECP2-R GGCCTCGGCGGCAGCGGCTG
Mulatta-MECP2-F TCTGACATTGCTATGGAGAG
Mulatta-MECP2-R CATAAGGAGAAGAGACAACAG
Mouse-Mecp2-F TTCTGAGCAAGCTGTAACA
Mouse-Mecp2-R ACCTGAACACCTTCTGATG
表2  人、猴MECP2、鼠Mecp2基因3号外显子引物
图1  sgRNA质粒构建
图2  在293T细胞上验证sgRNA效率
组合(mRNA) 组合浓度/(ng/μl) 编辑胚胎数 胚胎发育情况(8d)/囊胚率
sgRNA Target 1 20 10 Blastocyst(6);8-cell(2)2-cell(2)/60%
sgRNA Target 1 50 10 Blastocyst(5);4-cell(2)2-cell(3)/50%
BE3 20 10 Blastocyst(1);16-cell(2); 8-cell(3);2-cell(4)/10%
BE4max 20 10 Blastocyst(4);16-cell(3); 8-cell(3)/40%
Control 5 Blastocyst(5)/100%
表3  统计体外转录获得的mRNA注射到猕猴胚胎中的发育情况
组合(mRNA) 组合浓度/(ng/μl) 编辑胚胎数 胚胎发育情况(8d)/囊胚率
BE3∶sgRNA Target 1 20∶10 12 8-cell(1);4-cell(5);2-cell(4);death(2)/0%
BE4max∶sgRNA Target 1 20∶10 12 Blastocyst(2);4-cell(6);death(4)/16.7%
BE4max∶sgRNA Target 1 20∶10 11 Blastocyst(1);4-cell(4);3-cell(1);2-cell(5)/9%
BE4max∶sgRNA Target 1 20∶20 12 4-cell(5);2-cell(7)/0%
表4  BE3与BE4max在编辑胚胎的发育率统计
注射浓度/(ng/μl) 注射卵数/(囊胚发育情况) 胚胎突变个数(突变率)
第一组 20∶10 15/(9) 2(20%,80%)
50∶20 15/(8) 2(100%,5%)
100∶50 15/(5) 3(100%,60%,20%)
200∶100 15/(2) 2(100%,70%)
第二组 20:10 10/(8) 0
50∶20 12/(5) 1(70%)
100∶50 12/(5) 3(100%,80%,20%)
200∶100 10/(1) 2(90%,60%)
第三组 20∶10 30/(19) 3(20%,15%,15%)
50∶20 30/(11) 4(60%,20%,10%,5%)
100∶50 30/(9) 6(100%,100%,50%,40%,30%,20%)
200∶100 30/(4) 5(100%,100%,100%,60%,30%)
表5  BE4max系统编辑小鼠胚胎对发育率和突变率的统计
图3  小鼠胚胎验证
图4  猕猴胚胎检测
[1] Breakthrough of the year. The runners-up. Science, 2012,338(6114):1525-1532.
[2] Listed N A . Genetic microsurgery for the masses. Science, 2013,34(6165):1434-1435.
[3] John T . Making the cut. Science News, 2015,350(6267):1456-1457.
[4] Barrangou, Rodolphe . RNA-mediated programmable DNA cleavage. Nature Biotechnology, 2012,30(9):836-838.
[5] Cox D B T, Platt R J, Zhang F . Therapeutic genome editing:prospects and challenges. Nature Medicine, 2015,21(2):121-131.
[6] Komor A C, Kim Y B, Packer M S , et al. Programmable editing of a target base kkkin genomic DNA without double-stranded DNA cleavage. Nature, 2016.
[7] Yu W, Xiaohui Z, Dali L . The “new favorite” of gene editing technology-single base editors. Hereditas(Beijing), 2017,39(12).
[8] Kim K, Ryu S M, Kim S T , et al. Highly efficient RNA-guided base editing in mouse embryos. Nature Biotechnology, 2017.
doi: 10.1038/s41587-020-0484-5 pmid: 32518402
[9] Liu Z, Chen M, Chen S , et al. Highly efficient RNA-guided base editing in rabbit. Nat Commun, 2018,9(1):2717.
[10] Zhi quan, Liu, Mao , et al. Expanded targeting scope and enhanced base editing efficiency in rabbit using optimized xCas9(3.7). Cellular and Molecular Life Sciences, 2019.
[11] Liu Z, Shan H, Chen S , et al. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion. Faseb J, 2019,33(8):9210-9219.
[12] Chahrour M, Zoghbi H . The story of Rett syndrome: from clinic to neurobiology. Neuron, 2007,56(3):422-437.
pmid: 17988628
[13] Laurvick C L, Klerk N D, Bower C , et al. Rett syndrome in Australia: a review of the epidemiology. Journal of Pediatrics, 2006,148(3):0-352.
[14] Neul J L, Fang P, Barrish J , et al. Specific mutations in Methyl-CpG-Binding Protein 2 confer different severity in Rett syndrome. Neurology, 2008,70(16):1313-1321.
doi: 10.1212/01.wnl.0000291011.54508.aa pmid: 18337588
[15] Glaze D G, Percy A K, Skinner S , et al. Epilepsy and the natural history of Rett syndrome. Neurology, 2010,74(11):909-12.
pmid: 20231667
[16] Guy J, Hendrich B, Holmes M , et al. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nature Genetics, 2001,27(3):322.
pmid: 11242117
[17] Chen Y, Zheng Y, Kang Y , et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Human Molecular Genetics, 2015: ddv120.
[18] Niu Y, Shen B, Cui Y , et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 2014,156(4):836-843.
[19] Chen Y, Yu J, Niu Y , et al. Modeling Rett syndrome using TALEN-edited MECP2 mutant cynomolgus monkeys. Cell, 2017,169(3):945-955.
[20] Puping Liang, Hongwei Sun, Ying Sun , et al. Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein & Cell, 2017,8(8).
[21] Liang P, Ding C, Sun H , et al. Correction of β-thalassemia mutant by base editor in human embryos. Protein & Cell, 2017,8(11).
doi: 10.1007/s13238-017-0381-y pmid: 28224292
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[2] 王玥,牟彦双,刘忠华. 基于CRISPR/Cas系统的单碱基编辑技术研究进展*[J]. 中国生物工程杂志, 2020, 40(12): 58-66.
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