CN108823249A - CRISPR/Cas9构建notch1a突变体斑马鱼的方法 - Google Patents

CRISPR/Cas9构建notch1a突变体斑马鱼的方法 Download PDF

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CN108823249A
CN108823249A CN201810527941.9A CN201810527941A CN108823249A CN 108823249 A CN108823249 A CN 108823249A CN 201810527941 A CN201810527941 A CN 201810527941A CN 108823249 A CN108823249 A CN 108823249A
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zebra fish
notchla
notch1a
mutant
grna
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张庆华
季策
董雪红
张秋月
岳倩文
李伟明
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Shanghai Maritime University
Shanghai Ocean University
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Abstract

本发明公开了涉及一种CRISPR/Cas9构建notch1a突变体斑马鱼的方法；包括如下步骤：确定notch1a基因敲除的靶点位置；以pUC19‑gRNA scaffold质粒为模板，使用引物T7‑notch1a‑sfd、tracr rev进行PCR扩增；PCR产物纯化、体外转录获得gRNA；将gRNA与Cas9mRNA导入斑马鱼胚胎中，培养获得稳定遗传的notch1a突变体。本发明利用CRISPR/Cas9技术，通过选择一段独特的打靶区，使得斑马鱼中的notch1a被敲除，又不“误伤”其他基因，形成notch1a基因敲除的斑马鱼；另外，本发明还公开了notch1a基因缺失斑马鱼突变体的表型，对于研究Notch信号通路中Notch1a受体的作用意义重大。

Description

CRISPR/Cas9构建notch1a突变体斑马鱼的方法
技术领域
本发明涉及一种斑马鱼突变体，具体涉及一种通过CRISPR/Cas9技术制备notch1a突变体斑马鱼的方法及突变体表型的确定。
背景技术
Notch信号通路被认为是在早期发育过程中以及多种细胞的功能行使中扮演着重要角色的通路。其主要作用包括细胞的增殖、分化以及干细胞的更新等。Notch受体高度保守，其功能的行使依赖于出现在邻近细胞上的特异性配体。在机体的发育过程、肿瘤细胞的形成及增殖过程和遗传病的调控方面Notch信号通路都发挥着重要作用。在神经系统中，Notch信号通路也具有广泛的调控作用，影响神经干细胞的分化及数量，同时还可以调节多种细胞的形成。
CRISPR/Cas(Clustered Regularly Interspersed Short PalindromicRepeats，CRISPR/CRISPR-associated genes，Cas gene)系统是一种微生物的后天免疫系统，它利用向导RNA核酸酶对外源基因进行切割。CRISPR/Cas有三种类型：I型、II型和III型，其中II型只需要一个Cas9核酸内切酶切割DNA双链，即CRISPR/Cas9系统。该系统由Cas9核酸酶连同两种非编码性RNA，即CRISPR RNA(crRNA)和反式激活crRNA(trans-activatingcrRNA)共同组成。相比于其它的基因编辑技术，如ZFNs和TALENs，CRISPR/Cas9系统具有容易合成，打靶效率高和同时编辑多个基因的优势。CRISPR/Cas9系统的高效性不但确保了在体细胞内对基因进行突变，同时也能造成生殖细胞的突变，从而将突变基因传递到下一代。由于斑马鱼发育快，该系统对基因读码框序列破坏的高效性使得我们可以在短时间内建立稳定的可遗传的突变斑马鱼品系，这将有利于推动基因功能的研究。
申请人发现在斑马鱼中Notch1a与哺乳动物中NOTCH1具有较高的相似性。利用基因敲除的突变体可以有效的研究Notch通路的相关功能。目前关于小鼠中Notch1突变体的研究不足，并且小鼠模型的构建及维护费用昂贵。而在斑马鱼上使用morpholino敲降notch1a并不能得到体节及节间血管紊乱的表型。
斑马鱼notch1a基因位于21号染色体上，有3个转录本，其中最长的一个转录本mRNA全长7474bp，编码2438个氨基酸，含有33个外显子和32个内含子。如何选择一个有功能的靶点，使整个基因失去功能而且出现易于筛选的表型是十分困难的，犹如大海捞针。良好的打靶区的选择对于突变体的制备至关重要。并且，成功构建notch1a的突变体，并以此为模型研究早期发育中Notch通路的功能是十分必要的。
发明内容
本发明的目的在于提供一种利用CRISPR/Cas9构建notch1a突变体斑马鱼的方法及突变体表型的确定。本发明利用CRISPR/Cas9技术选取第16个外显子上特异的打靶位点构建了notch1a的突变体。随机突变的结果共得到4种突变类型，分别为在靶点附近缺失4bp、10bp、19bp和31bp。对突变的序列进行氨基酸翻译发现均会形成蛋白翻译的提前终止，缺失4bp突变体会在947位氨基酸处出现翻译终止；缺失10bp突变体会在945位氨基酸处出现翻译终止；缺失19bp突变体会在942位氨基酸处出现翻译终止；缺失31bp突变体会在938位氨基酸处出现翻译终止。突变体会出现体节和节间血管紊乱的表型，而且这4种不同的突变体出现一致的表型特征。
本发明的目的是通过以下技术方案来实现的：
本发明涉及一种斑马鱼notch1a基因突变体的制备方法，所述方法包括如下步骤：
S1、确定notch1a敲除的靶点在斑马鱼notch1a的基因序列的第16个外显子上；
S2、根据步骤S1确定的靶点序列设计扩增引物；
S3、以pUC19-gRNA scaffold质粒为模板，使用引物T7-notch1a-sfd、tracr rev进行PCR扩增；
S4、对步骤S3的PCR产物进行体外转录，纯化获得gRNA；
S5、以pXT7-hCas9质粒为模板，体外转录合成Cas9 mRNA；
S6、将gRNA与Cas9 mRNA导入斑马鱼一细胞期胚胎中；
S7、培养获得稳定遗传的斑马鱼notch1a突变体。
优选的，步骤S1中，所述靶点序列为GGAGTGTGTGAAAACCTGCG(SEQ ID NO.1)。
优选的，步骤S2中，所述扩增引物为notch1a F：CGTGTGAGGTGGACATTA(SEQ IDNO.4)；notch1a R：CATTAGTTAAGTGAGGTGTGAG(SEQ ID NO.5)。
优选的，步骤S3中，所述引物T7-notch1a-sfd的序列为TAATACGACTCACTATAGGAGTGTGTGAAAACCTGCGGTTTTAGAGCTAGAAATAGC(SEQ ID NO.2)。
优选的，步骤S3中，所述引物tracr rev的序列为AAAAAAAGCACCGACTCGGTGCCAC(SEQ ID NO.3)。
优选的，步骤S4中，所述gRNA的序列为T7启动子+靶位点+pUC19-gRNA固定序列，是使用T7-notch1a-sfd和tracr rev引物对，以pMD19-gRNA scaffold质粒为模板，使用高保真酶High-Fidelity PCR Master Mix with HF Buffer，电泳、切胶回收获得。
优选的，步骤S5中，所述Cas9 mRNA是通过包括如下步骤的方法制备而得：
A1、pXT7-hCas9质粒线性化，用XbaI内切酶酶切质粒pXT7-hcas9；
A2、酶切产物纯化，用DNA Clean&Contentrator TM-5kit纯化试剂盒对上述酶切产物进行纯化；
A3、体外转录Cas9 mRNA，用mMESSAGE mMACHINE T7 ULTRA kit体外转录试剂盒对Cas9 mRNA进行体外转录；
A4、对得到的产物进行加尾后用Nanodrop 2000C测浓度，并-80℃保存备用。
优选的，步骤S6中，将gRNA与Cas9 mRNA导入斑马鱼，具体为：将gRNA与Cas9 mRNA混合，显微注射到斑马鱼一细胞期胚胎中；其中，gRNA终浓度为100ng/μL，Cas9 mRNA终浓度为400ng/μL。
优选的，步骤S7具体包括如下步骤：
B1、对导入gRNA与Cas9 mRNA的斑马鱼进行notch1a敲除检测，确定notch1a F0靶点突变效率；
B2、将notch1a F0成鱼与野生型斑马鱼外交，得到F1胚胎；经基因型鉴定获得notch1a F1突变体斑马鱼；
B3、将相同突变的notch1a F1突变体斑马鱼内交，获得notch1a F2突变体斑马鱼；
B4、鉴定为F2中notch1a敲除的纯合子具有纯合致死的现象，F2中notch1a基因敲除的杂合子即所述稳定遗传的斑马鱼notch1a突变体。
优选的，步骤B1中，notch1a敲除检测采用的引物序列为notch1a F：CGTGTGAGGTGGACATTA(SEQ ID NO.4)；notch1a R：CATTAGTTAAGTGAGGTGTGAG(SEQ IDNO.5)。
与现有技术相比，本发明具有如下有益效果：
1、利用CRISPR/Cas9技术及一段特异的打靶位点，首次在斑马鱼中敲除notch1a。
2、notch1a突变可稳定遗传，方便深入研究notch1a的基因功能。
3、notch1a突变体具有明显体节紊乱及节间血管紊乱表型。
附图说明
图1为notch1a F0敲除检测示意图；其中，a为notch1a F0斑马鱼胚胎PCR产物，b为T7E1内切酶酶切鉴定结果，c为PCR产物测序结果；
图2为notch1a F1突变类型统计；
图3为notch1a F2缺失31bp纯合突变体表型鉴定；
图4为notch1a F2四种不同突变类型纯合突变体的表型鉴定；
图5为notch1a突变体节间血管紊乱表型。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例
1材料及设备
1.1实验用鱼
本实验中所用的斑马鱼均为AB品系，购置于中国科学院上海生命科学研究院生物化学与细胞生物学研究所斑马鱼平台。
1.2质粒
pXT7-hCas9质粒，pUC19-gRNA scaffold质粒来源于文献：Chang N，Sun C，Gao L，Zhu D，Xu X，Zhu X，Xiong JW，Xi JJ.Genome editing with RNA-guided Cas9 nucleasein zebrafish embryos，CellRes，2013，23(4)：465-472。
1.3主要试剂
DNA Clean&Contentrator-5(ZYMO RESEARCH，D4004)，普通DNA纯化试剂盒(TIANGEN，DP204-03)，T7in vitro Transcription Kit(Ambion，AM1314)，乙醇(无水乙醇)(国药集团化学试剂有限公司，10009218)，GenCrisprNLS-Cas9-NLS(金斯瑞，Z03389-25)，Premix TaqTM(Ex TaqTM Version 2.0plus dye)(TAKARA，RR902)，DNA MarkerI(TIANGEN，MD101-02)，T7endonuclease 1(NEW ENGLANDInc.，M0302L)，快速质粒小提试剂盒(TIANGEN，DP105)，DH5a感受态细胞(天根生化科技有限公司，CB101-03)，LBBroth(上海生工，D915KA6602)，LB Broth agar(上海生工，D911KA6566)，pMDTM19-TVectorCloning Kit(TAKARA，6013)。
1.4主要仪器
PCR仪(品牌：BIO-RAD，型号：c1000TouchTMThermal Cycler)，离心机(品牌：Eppendorf，型号：Centrifuge 5424)，震荡混匀仪(品牌：VORTEX-GENIE，型号：G560E)，分光光度计(品牌：Thermo Scientific，型号：Nanodrop 2000C)，电泳仪(品牌：BIO-RAD，型号：PowerPac Basic)，照胶仪(品牌：BIO-RAD，型号：Gel Doc EZ Imager)，电子天平(品牌：METTLER TOLEDO，型号：AL104)，玻璃毛细管(品牌：WPI，型号：TW100F-4)，纯水仪(品牌：Millipore，型号：Milli-Q Direct 8)，垂直拉针仪(品牌：NARISHIGE，型号：PC-10)，恒温摇床(品牌：Innova，型号：40R)，磨针器(品牌：NARISHIGE，型号：EG-400)，显微操作仪(品牌：Warner Instruments，型号：PL1-100A Plus)，恒温水浴锅(品牌：精宏，型号：H1401438，DK-8D)，4℃冰箱(品牌：Haier，型号：HYC-610)，-40℃低温冰箱(品牌：Haier，型号：DW-40L508)，-80℃超低温冰箱(品牌：Panasonic，型号：MDF-U53V)，高压蒸汽灭菌锅(品牌：SANYO，型号：MLS-3780)。
2实验方法
2.1gRNA合成
(1)靶点设计
a、查找序列：在Ensemb1数据库查找并下载斑马鱼notch1a的基因序列。
b、靶点设计：利用http：//zifit.partners.org/ZiFiT/ChoiceMenu.aspx网站在notch1a第16个外显子序列上设计靶点，如表1，具体靶点序列为：GGAGTGTGTGAAAACCTGCG(SEQ ID NO.1)。
表1 notch1a基因靶位点序列
c、靶点特异性检测：在NCBI网站将设计的靶点序列通过Blast比对，验证靶位点特异性。
d、亲本检测：对用于基因敲除的野生型斑马鱼进行剪尾提取基因组DNA，PCR扩增靶点及附近序列。
e、酶切检测：用T7E1内切酶酶切检测用于基因敲除的野生型斑马鱼靶点附近序列。
f、测序鉴定：将PCR产物测序，峰图及序列比对，使用该区域序列一致的野生型斑马鱼作为亲本。
(2)设计检测引物：引物距离靶点两侧大于100bp，并且上游引物到靶点的距离与下游引物到靶点的距离相差大于100bp，扩增片段约500bp(表2)。
表2实验所用引物信息
gRNA产物合成：以pUC19-gRNA scaffold质粒为模板，使用引物T7-notch1a-sfd、tracr rev和2×EasyTaq PCR Super Mix(+dye)扩增片段并用试剂盒纯化。(4)体外转录：
表3反应体系
Nuclease-free Water to 20μL
DNA Template 1μg
10×Transcription Buffer 2μL
10mM ATP 1μL
10mM CTP 1μL
10mM GTP 1μL
10mM UTP 1μL
T7Enzyme Mix 2μL
(最后添加10×Transcription Buffer和T7Enzyme mix)
混匀并短暂离心后，37℃孵育80min；之后向体系中加入1μL TURBO DNase并混匀，短暂离心后37℃孵育15min。
(5)纯化gRNA：
a、向20μL体外转录体系中加入2.5μL 4M的LiCl和100μL无水乙醇，混匀并短暂离心后放于-80℃冰箱至少1h。
b、从冰箱取出后，4℃，12000rmp，离心15min。弃上清后用70％乙醇清洗沉淀。4℃，8000rmp，离心5min。弃上清后将离心管放于通风橱中使乙醇挥发干净。
c、加入10μL DEPC水溶解gRNA沉淀。
d、用Nanodrop 2000C检测浓度。
2.2显微注射
将gRNA与Cas9 mRNA混合，利用显微注射仪注射到斑马鱼一细胞期胚胎中。混合注射终浓度：gRNA为100ng/μL，Cas9 mRNA为400ng/μL。
2.3T7E1酶切检测敲除效率
a、提取胚胎基因组
每组5枚胚胎，加35μL的50mM NaOH，95℃孵育20min，中间取出振荡。之后加3.5μL1M Tris·HCl(pH≈8.0)，振荡混匀后离心。
b、PCR扩增目的片段
使用表里中notchla F(SEQ ID NO.4)与notch1a R(SEQ ID NO.5)引物扩增目的片段。
c、T7E1内切酶酶切检测
表4 T7E1酶切体系
H2O to 10μL
PCR产物 5μL
Buffer 1.1μL
95℃孵育5min，冷却至室温，加0.25μL T7E1酶，37℃孵育45min。
d、电泳检测及敲除效率检测
电泳后利用凝胶电泳成像仪对电泳的琼脂糖凝胶成像，并计算敲除效率。
2.4notch1a F2纯合突变体斑马鱼表型检测
对notch1a F2斑马鱼胚胎进行拍照观察，并统计出现表型胚胎数量。
2.5不同突变类型表型统计
对不同缺失类型的斑马鱼F2胚胎进行表型统计与基因型鉴定。
3实验结果
3.1notch1a突变体的构建
3.1.1notch1a F0基因敲除检测结果
结果显示notchla基因敲除成功，利用Image Lab 5.1软件计算敲除效率达到40％以上。测序峰图显示在20bp靶点处出现套峰，证明敲除成功(图1)。
3.1.2notchlaF1突变体斑马鱼检测
对F1斑马鱼进行基因型检测，共得到4种突变类型，分别为在靶点附近缺失4bp、10bp、19bp和31bp。对突变的序列进行氨基酸翻译发现均会形成翻译的提前终止(图2)。notch1a可编码2438个氨基酸，缺失4bp突变体会在947位氨基酸处出现翻译终止；缺失10bp突变体会在945位氨基酸处出现翻译终止；缺失19bp突变体会在942位氨基酸处出现翻译终止；缺失31bp突变体会在938位氨基酸处出现翻译终止。
3.1.3notch1a F2突变体斑马鱼检测
对F2斑马鱼进行统计发现notch1a突变纯合致死，具体死亡时间为10-13dpf(表5)。
表5 notch1a纯合子死亡统计
大小 总数 死亡数 比例
10dpf 116 23 19.8％
11dpf 116 33 28.4％
12dpf 116 45 38.8％
13dpf 116 15 13.0％
3.1.4notch1a F2突变体表型鉴定
表型鉴定结果显示，notch1a突变纯合子在第5-7个体节之后会出现体节边界紊乱的表型(图3)。并且对不同突变类型纯合子进行鉴定发现，四种不同突变类型纯合子出现相一致表型(图4)；并且notch1a突变纯合子还具有节间血管生长紊乱的表型(图5)。
序列表
<110> 上海海洋大学
<120> CRISPR/Cas9构建notch1a突变体斑马鱼的方法
<130> 2018
<141> 2018-05-28
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Danio rerio
<400> 1
ggagtgtgtg aaaacctgcg 20
<210> 2
<211> 57
<212> DNA
<213> Artificial sequence
<220>

Claims (8)

1.一种斑马鱼notchla突变体的制备方法，其特征在于，所述方法包括如下步骤：
S1、确定notchla基因敲除的靶点在斑马鱼notchla的基因序列的第16个外显子上；
S2、根据步骤S1确定的靶点序列设计扩增引物；
S3、以pUC19-gRNA scaffold质粒为模板，使用引物T7-notchla-sfd、tracr rev进行PCR扩增；
S4、对步骤S3的PCR产物进行体外转录，纯化获得gRNA；
S5、以pXT7-hCas9质粒为模板，体外转录合成Cas9 mRNA；
S6、将gRNA与Cas9 mRNA导入斑马鱼一细胞期胚胎中；
S7、培养获得稳定遗传的斑马鱼notchla突变体。
2.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S2中，所述靶点序列为SEQ ID NO.1所示的序列。
3.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S3中，所述引物T7-notchla-sfd的序列为SEQ ID NO.2所示的序列。
4.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S3中，所述引物tracr rev的序列为SEQ ID NO.3所示的序列。
5.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S5中，所述Cas9 mRNA是通过包括如下步骤的方法制备而得：
A1、pXT7-hCas9质粒线性化，用XbaI内切酶酶切质粒pXT7-hCas9；
A2、酶切产物纯化，用DNA Clean&Contentrator TM-5 kit纯化试剂盒对酶切产物进行纯化；
A3、体外转录Cas9 mRNA，用mMESSAGE mMACHINE T7 ULTRA kit体外转录试剂盒对Cas9mRNA进行体外转录；
A4、对得到的产物进行加尾后用Nanodrop 2000C测浓度并-80℃保存备用。
6.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S6中，将gRNA与Cas9 mRNA导入斑马鱼，具体为：将gRNA与Cas9 mRNA混合，显微注射到斑马鱼一细胞期胚胎中；其中，gRNA终浓度为100ng/μL，Cas9 mRNA终浓度为400ng/μL。
7.根据权利要求1所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤S7具体包括如下步骤：
B1、对导入gRNA与Cas9 mRNA的斑马鱼进行notchla敲除检测，确定notchla F0靶点突变效率；
B2、将notchla F0成鱼与野生型斑马鱼外交，得到F1胚胎；经基因型鉴定获得notchlaF1突变体斑马鱼；
B3、将相同突变的notchla F1突变体斑马鱼内交，获得notchla F2突变体斑马鱼；
B4、鉴定为F2中notchla敲除的纯合子具有纯合致死的现象，F2中notchla敲除的杂合子即所述稳定遗传的斑马鱼notchla突变体。
8.根据权利要求7所述的斑马鱼notchla突变体的制备方法，其特征在于，步骤B1中，notchla敲除检测采用的引物序列为如SEQ ID NO.4和SEQ ID NO.5所示的序列。
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