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CN107099533A - 一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用 - Google Patents

一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用 Download PDF

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CN107099533A
CN107099533A CN201710483275.9A CN201710483275A CN107099533A CN 107099533 A CN107099533 A CN 107099533A CN 201710483275 A CN201710483275 A CN 201710483275A CN 107099533 A CN107099533 A CN 107099533A
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牟彦双
刘忠华
尹智
翁晓刚
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Abstract

一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用，涉及一种sgRNA导向序列及应用。该sgRNA导向序列为CGTCTCGCGCTTGGACTCAG。本发明提供的sgRNA导向序列可通过CRISPR/Cas9系统敲除或编辑IGFBP3基因，进而消除IGFBP3的表达，为制备IGFBP3转基因猪奠定基础。本发明应用于基因工程领域。

Description

一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用
技术领域
本发明涉及一种sgRNA导向序列及应用。
背景技术
CRISPR/cas9基因编辑系统是基于II类CRISPR/Cas系统改造而来的。Cas9蛋白是唯一所需要的，用来介导外源DNA沉默的Cas蛋白。2012年，Jinek通过体外实验证实Cas9蛋白对原间隔序列进行切割不仅仅需要与原间隔序列互补的成熟的crRNA，还需要tracrRNA；通过对切割后的片段测序发现，tracrRNA：crRNA指导Cas9蛋白切割位点是具有位点特异性的，无论是环状质粒还是线性DNA片段，切割位点均在PAM序列上游的3bp处；Cas9蛋白包含HNH及RuvC内切酶结构域，无论对哪个结构域进行突变，均无法有效的进行切割，只是在双链DNA上产生切口，实验证实HNH结构域会对与crRNA互补链进行切割，而RuvC结构域则会对另一条链进行切割；对于众多的CRISPR/Cas系统，识别自我与非我的关键就在于保守的PAM序列，对于II类CRISPR/Cas系统，PAM序列为NGG，当对PAM序列进行突变后，将无法进行有效的切割；此外，还成功的将crRNA与tracrRNA两条链成功的融合成一条大约100nt的单链向导RNA(guideRNA，gRNA)，并能正常发挥crRNA：tracrRNA的功能。这项工作为CRISPR/Cas系统由细菌的天然防御系统向基因编辑工具的转变奠定了基础。2013年1月，麻省理工学院以及哈佛医学院同时再science上首次发表了使用CRISPR/Cas系统对哺乳动物细胞进行基因编辑。在麻省理工学院的研究中，使用酿脓链球菌的II类CRISPR/Cas系统，针对人的Emx1位点设计的crRNA能够高效的在人的293FT细胞基因组的Emx1位点上产生突变。针对该基因的不同PAM序列设计crRNA以及crRNA与tracrRNA嵌合在一起的chiRNA，对细胞进行基因编辑，crRNA均能产生有效的突变，但可能由于RNA的二级结构的影响，并不是所有的chiRNA都能进行高效的位点突变，此外，利用该系统对基因组进行切割后，不仅能够通过非同源性末端接合(NHEJ)的方式进行修复，获得在切割位点附近的碱基缺失或是插入，还可以通过同源重组修复(HDR)的方式，实现特异基因片段的重组，此外，通过针对同一基因的两个位点设计crRNA，可以成功的进行一段片段的基因敲除。使用RuvC结构域突变的Cas9蛋白也能够通过同源重组的修复方式进行修复，但无法获得非同源性末端接合的突变。哈佛医学院也获得了类似的结果，且CRISPR/Cas9系统的基因编辑效率与先前出现的TALEN效率类似，并能够同时针对多个位点进行基因编辑。进一步证实了CRISPR/Cas9系统终将成为真核生物基因编辑领域强大的基因编辑工具。
Insulin like growth factorbinding protein 3(IGFBP3)基因属于生长激素超家族基因。主要作用是与IGF1基因和IGF2结合使IGF1和IGF2基因在体内能正常运输。IGFBP3基因主要表达组织：早期胚胎，内胚层，间充质细胞，消化道系统，心血管系统，淋巴系统，肝胆系统，骨骼肌，神经系统，生殖系统，泌尿系统。IGFBP3基因单敲除后小鼠表行没有变化，IGFBP3基因双敲除后，主要表型为白色脂肪组织减少，体重增加，肝脏重增加，肌肉重量增加。同时，对高糖和高油脂食物出现耐受。这些表型的出现一般认为是IGFBP3基因敲除后，IGFBP2基因出现补偿效应，IGFBP2基因的表达量升高，导致IGF1基因在血液中含量增高，最终导致小鼠体重的增加。同时，基因敲除小鼠的采食量相比于正常小鼠没有发生显著性改变。在猪中研究表明，IGFBP3基因与猪屠体重和背膘厚呈显著相关。
发明内容
本发明目的在于提供一种特异靶向猪IGFBP3基因的sgRNA导向序列及其应用，该sgRNA导向序列可以用于敲除猪IGFBP3基因，为制备转基因猪奠定基础。
本发明特异靶向猪IGFBP3基因的sgRNA导向序列为IGFBP3-SgRNA1，其核苷酸序列为：5’-CGTCTCGCGCTTGGACTCAG-3’，如SEQ ID NO：1所示，位于基因IGFBP3第二外显子。
上述特异靶向猪IGFBP3基因的sgRNA导向序列在敲除猪IGFBP3基因中的应用，具体方法如下：
一、在猪IGFBP3基因的sgRNA导向序列的5’端加上CACC得到正向寡核苷酸；同时根据导向序列获得得其对应的DNA互补链，并且再起5’端加上AAAC得到反向寡核苷酸；分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；
二、将步骤一制得的双链DNA与CRISPR/Cas9载体连接，得到重组敲除表达载体；
三、将步骤二制得的重组表达载体连接入药物筛选抗性基因序列；
四、将步骤三制得的重组敲除表达载体转染细胞，筛选稳定转染细胞，得到成功敲除IGFBP3基因的细胞。
进一步的，步骤二中所述的CRISPR/Cas9载体为pX330载体。
进一步的，步骤三中所述的药物筛选抗性基因序列为G418抗性基因序列。
进一步的，步骤四中所述转染细胞的方法为脂质体转染法。
进一步的，步骤四中所述的细胞为猪成纤维细胞。
上述特异靶向猪IGFBP3基因的sgRNA导向序列在特异识别和靶向修饰猪IGFBP3基因中的应用。
上述特异靶向猪IGFBP3基因的sgRNA导向序列在构建猪IGFBP3基因突变库中的应用。
特异靶向猪IGFBP3基因的sgRNA导向序列，能产生基因插入突变、基因序列置换、基因序列缺失等多种类型IGFBP3基因突变体，可以在构建猪IGFBP3基因突变库中的应用。
本发明相对于现有技术具有如下的优点及效果：
本发明根据sgRNA导向序列设计合成两条单链核苷酸序列，退火形成双链，然后与Cas9载体连接，利用Cas9载体将sgRNA及CRISPR系统引入目标细胞中，Cas9蛋白会在sgRNA的引导下找到与其匹配的基因组DNA序列，进行剪切，实现猪基因IGFBP3的敲除。
(1)利用质粒载体pX330将sgRNA以及CRISPR系统引入细胞中，Cas9蛋白会在sgRNA的引导下找到与其匹配的DNA序列，进行剪切。质粒载体安全性高，不会引起细胞的免疫反应。
(2)载体中含有G418抗性基因，利用G418对细胞进行筛选，未转入pX330载体的细胞将在筛选过程中被淘汰。
(3)本发明提供的特异靶向猪IGFBP3基因的sgRNA导向序列，可通过CRISPR/Cas9系统敲除或编辑IGFBP3基因，进而消除IGFBP3的表达，为制备IGFBP3转基因猪奠定基础。
附图说明
图1是特异靶向猪IGFBP3基因的sgRNA导向序列及其位置示意图。
图2是PCR扩增猪IGFBP3基因外显子2片段电泳图。
具体实施方式
下面对本发明的实施例做详细说明，以下实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方案和具体的操作过程，但本发明的保护范围不限于下述的实施例。
以下实施例中所使用的细胞为原代培养猪成纤维细胞，pX330，pEGFP-C1载体购自Addgene，内切酶BbsI，BsmBI购自NEB，G418和细胞培养基购自Sigma。
实施例1：
(1)sgRNA设计
根据猪IGFBP3基因的基因组序列(gene ID：001005156)，设计1个靶向猪IGFBP3基因的sgRNA。20nt的寡核苷酸sgRNA导向序列为：IGFBP3-SgRNA1:5’-CGTCTCGCGCTTGGACTCAG-3’，位于基因IGFBP3第二外显子；在其5’端加上CACC得到正向寡核苷酸序列；根据导向序列获得其对应的DNA互补链，并且在其5’端加上AAAC得到反向寡核苷酸。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的正向和反向sgRNA寡核苷酸序列：95℃变性5min，72℃退火10min；退火后可以形成带有粘性末端的双链DNA，具体寡核苷酸序列见表1。
表1sgRNA导向序列的寡核苷酸序列
核苷酸序列(5’至3’)
Sg1-F CACCCGTCTCGCGCTTGGACTCAG
Sg1-R AAACCTGAGTCCAAGCGCGAGACG
(2)构建表达sgRNA的载体
质粒载体pX330有BbsI酶切位点，用BbsI酶切，其中，酶切体系为10μL体系：BbsI1μL；10×NE Buffer 1μL；质粒2μL；ddH2O 6μL；酶切条件为：37℃酶切过夜；
将酶切后载体pX330与步骤(1)制得的退火双链利用T4连接酶进行连接，连接体系为10μL体系：pX330载体2μL，退火双链sgRNA 6μL，10NEB T4DNALigase Buffer 1μL，T4Ligase 1μL；连接条件为16℃连接过夜；
将连接产物转化感受态细菌DH5α，具体转化方法为：-80℃取出感受态细菌DH5α，在冰浴溶解；然后100μL感受态细菌中加入10μL的上述连接产物，混匀后冰浴30min；42℃水浴100s热激活，冰浴2min；然后加入900μL SOC培养基，37℃摇床60min；涂AMP+(100μg/mL)固体SOC培养基平板，37℃培养过夜，挑取阳性克隆，在SOC液体培养基中37℃摇床过夜，之后用Tiangen质粒抽提试剂盒提取质粒并测序验证，得到表达sgRNA的pX330-IGFBP3-Sg1质粒载体。
实施例2：
(1)G418抗性基因的获得
根据质粒pEGFP-C1载体中G418抗性基因设计引物，引物寡核苷酸序列5’端加入BsmBI酶切位点，Kana-F：5-GGCGTCTCCGGGATGTGCGCGGAACCCCTATTTG-3，Kana-R：5-GGCGTCTCGCGCGCAATCTAAAGTATATATGAGTAACC-3，分别合成上下游引物序列，以质粒pEGFP-C1为模板进行PCR扩增，PCR反应体系为50μL体系：pEGFP-C1质粒1μL，2×PCRMix 25μL，上下游引物各1μL，ddH2O 22μL。PCR反应条件为：94℃5min，94℃30s，60℃45s，72℃1min40s，共33个循环，之后72℃延伸10min。获得的DNA片段命名为Kana。
表2扩增Kana片段的引物序列
序列(5’至3’)
Kana-F GGCGTCTCCGGGATGTGCGCGGAACCCCTATTTG
Kana-R GGCGTCTCGCGCGCAATCTAAAGTATATATGAGTAACC
(2)构建含有G418抗性基因的载体质粒
将pX330-IGFBP3-Sg1质粒载体和Kana片段DNA分别用BsmBI酶切，酶切体系为20μL体系：BbsI 1μL；10×NE Buffer 2μL；质粒或DNA片段4μL；ddH2O 13μL；酶切条件为：37℃酶切过夜；将酶切后的pX330-IGFBP3-Sg1质粒载体和Kana片段利用T4连接酶进行连接，连接步骤同实施例1中步骤(2)，连接产物转化感受态细菌DH5α，转化步骤同实施例1中步骤(2)，得到连接G418抗性基因的pX330-G418-IGFBP3-Sg1质粒载体。
实施例3：
(1)猪成纤维细胞的培养
猪胎儿成纤维细胞分离培养方法参见文献：信吉阁；成文敏；潘伟荣；卿玉波；查星琴；富国文；魏红江；曾养志。猪胎儿成纤维细胞和耳皮肤成纤维细胞培养方法的研究，黑龙江畜牧兽医，2013(9)：175-179.
(2)质粒转染和阳性细胞筛选
将重组质粒pX330-G418-IGFBP3-Sg1通过脂质体转染的方法转染猪胎儿成纤维细胞，得到重组细胞。转染的具体步骤参见脂质体3000(Invitrogen，货号：11668019)操作说明书方法进行转染。
将转染后细胞利用1000ng/mL的G418对转染后的细胞进行筛选，筛选持续10d，对筛选后的细胞进行培养并冷冻保存。
(3)基因敲除效果鉴定
根据所设计的sgRNA序列设计鉴定引物，用于对敲除后目的片段进行鉴定，所设计的引物如表3所示：
表3扩增IGFBP3片段的引物序列
序列(5’至3’)
E2-F GTCGTTAAATGCCAATGACCCTC
E2-R AGGTGCACCCCACCCCACCAT
利用Takara公司TaKaRaMiniBEST Universal Genomic DNA Extraction KitVer.5.0，Code NO.9765.对稳定转染的细胞进行基因组抽提，利用鉴定引物进行PCR鉴定。
其PCR反应体系为50μL体系：基因组DNA 1μL，2×PCRMix 25μL，上下游引物各1μL，ddH2O 22μL。PCR反应条件为：94℃预变性5min，94℃变性30s，58℃退火30s，72℃延伸30s，共33个循环，之后72℃延伸10min。获得的DNA片段命名为IGFBP3-E2。
PCR反应产物琼脂糖凝胶电泳后，利用Gel Extraction Kit(Takara)进行胶回收，对胶回收产物进行测序分析，测序结果如图2所示。测序结果表明细胞基因组在基因编辑处发生了突变，对照组与野生型基因组进行了对比没有发生变化。本发明成功建立了敲除IGFBP3基因的猪成纤维细胞。
序列表
<110>东北农业大学
<120>一种特异靶向猪IGFBP3基因的sgRNA导向序列及应用
<160>7
<210>1
<211> 20
<212> DNA
<213>人工序列
<220>
<223> 导向序列IGFBP3-SgRNA1
<400>1
cgtctcgcgcttggactcag 20
<210>2
<211> 24
<212> DNA
<213>人工序列
<220>
<223> 寡核苷酸序列Sg1-F
<400>2
cacccgtctcgcgcttggactcag 24
<210>3
<211> 24
<212> DNA
<213>人工序列
<220>
<223> 寡核苷酸序列Sg1-R
<400>3
aaacctgagtccaagcgcgagacg 24
<210>4
<211>34
<212> DNA
<213>人工序列
<220>
<223> 序列Kana-F
<400>4
ggcgtctccgggatgtgcgcggaacccctatttg34
<210>5
<211>38
<212> DNA
<213>人工序列
<220>
<223> 序列Kana-R
<400>5
ggcgtctcgcgcgcaatctaaagtatatatgagtaacc38
<210>6
<211>23
<212> DNA
<213>人工序列
<220>
<223>引物序列E2-F
<400>6
gtcgttaaatgccaatgaccctc23
<210>7
<211>21
<212> DNA
<213>人工序列
<220>
<223>引物序列E2-R
<400>7
aggtgcaccccaccccaccat21

Claims (8)

1.一种特异靶向猪IGFBP3基因的sgRNA导向序列，其特征在于该序列为IGFBP3-SgRNA1，IGFBP3-SgRNA1的核苷酸序列如SEQ ID NO：1所示。
2.权利要求1所述的特异靶向猪IGFBP3基因的sgRNA导向序列在敲除猪IGFBP3基因中的应用，具体如下：
一、在猪IGFBP3基因的sgRNA导向序列的5’端加上CACC得到正向寡核苷酸；同时根据导向序列获得得其对应的DNA互补链，并且再起5’端加上AAAC得到反向寡核苷酸；分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；
二、将步骤一制得的双链DNA与CRISPR/Cas9载体连接，得到重组敲除表达载体；
三、将步骤二制得的重组表达载体连接入药物筛选抗性基因序列；
四、将步骤三制得的重组敲除表达载体转染细胞，筛选稳定转染细胞，得到成功敲除IGFBP3基因的细胞。
3.根据权利要求2所述的应用，其特征在于步骤二中所述的CRISPR/Cas9载体为pX330载体。
4.根据权利要求2所述的应用，其特征在于步骤三中所述的药物筛选抗性基因序列为G418抗性基因序列。
5.根据权利要求2所述的应用，其特征在于步骤四中所述转染细胞的方法为脂质体转染法。
6.根据权利要求2所述的应用，其特征在于步骤四中所述的细胞为猪成纤维细胞。
7.权利要求1所述的特异靶向猪IGFBP3基因的sgRNA导向序列在特异识别和靶向修饰猪IGFBP3基因中的应用。
8.权利要求1所述的特异靶向猪IGFBP3基因的sgRNA导向序列在构建猪IGFBP3基因突变库中的应用。
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