CN107119053A - 一种特异靶向猪MC4R基因的sgRNA导向序列及其应用 - Google Patents

一种特异靶向猪MC4R基因的sgRNA导向序列及其应用 Download PDF

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CN107119053A
CN107119053A CN201710483307.5A CN201710483307A CN107119053A CN 107119053 A CN107119053 A CN 107119053A CN 201710483307 A CN201710483307 A CN 201710483307A CN 107119053 A CN107119053 A CN 107119053A
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mc4r
pig
genes
sgrna targeting
targeting sequencings
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牟彦双
刘忠华
郭佳
马伟超
郭超
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Northeast Agricultural University
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Abstract

一种特异靶向猪MC4R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MC4R基因，进而消除MC4R的表达，为制备MC4R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MC4R基因的sgRNA导向序列及其应用
技术领域
本发明涉及一种sgRNA导向序列及应用。
背景技术
近十年来出现了一种新的研究手段，可以帮助科研人员对各种细胞和各种生物体内的几乎任意基因进行人工操作。这种新技术就是我们常说的“基因组编辑技术”，ZFN和TALEN都可以对DNA进行各种遗传修饰，这两种核酸酶的作用机制都是先对DNA双链分子进行切割，形成DNA双链断裂切口，然后激活细胞内的非同源末端连接修复机制，或者同源重组修复机制，利用细胞自身的修复机制对DNA进行遗传学修饰。而最新出现的CRISPR/Cas9基因编辑系统一经出现，便得到极大的发展及应用。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。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。
MC4R是第一个发现的与人类显性遗传疾病性肥胖相关的靶位点，Yeo等首次在两例早发性肥胖患者中发现了MC4R基因移码突变后，MC4R在人类能量和体重调节中的重要性逐渐地被揭示。人MC4R突变研究表明，其显性遗传多由于单体不足所导致，在极少数个体中也可出现隐性遗传的错义突变。MC4R基因突变属常染色体显性遗传，因此该基因具有表型的突变在人群中的发生率较高，迄今有近80例MC4R基因突变导致肥胖的病例报道，据估计BMI大于40的极度肥胖人群中有1％-4％是由于MC4R基因突变所致。在人类由MC4R基因突变引起的肥胖症其表现型多种多样，除了多食、肥胖外，并不伴发其他的内分泌代谢异常，甲状腺、肾上腺和生殖功能正常，不同于其他类型的单基因突变性肥胖。另外，MC4R基因多态性也可能与体脂分布及脂代谢相关，体型缺陷MC4R 导致的肥胖为青少年发病型，且女性的严重程度高于男性，其中大多数患者体脂分布有女性化倾向。在猪中对MC4R基因D298N突变位点进行了多态性分析，大白猪(引入品种)和北京黑猪(培育品种)含D298N位点等位突变所以瘦肉率高，肌内脂肪含量低。MC4R 基因D298N突变位点很可能与肌内脂肪含量相关。以上研究表明MC4R可以作为与猪脂肪沉积性状及肉质性状相关的候选基因。。
发明内容
本发明目的在于提供一种特异靶向猪MC4R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MC4R基因，为制备转基因猪奠定基础。
本发明特异靶向猪MC4R基因的sgRNA导向序列为MC4R-SgRNA1，其核苷酸序列为：5’-TGTGCAGTCCGTAGGTGCTG-3’，如SEQ ID NO：1所示，位于基因MC4R外显子。
上述特异靶向猪MC4R基因的sgRNA导向序列在敲除猪MC4R基因中的应用，具体方法如下：
一、在猪MC4R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，步骤三中所述转染细胞的方法为脂质体转染法。
进一步的，步骤三中所述筛选所用药物为嘌呤霉素。
进一步的，步骤三中所述的细胞为猪成纤维细胞。
上述特异靶向猪MC4R基因的sgRNA导向序列在特异识别和靶向修饰猪MC4R基因中的应用。
上述特异靶向猪MC4R基因的sgRNA导向序列在构建猪MC4R基因突变库中的应用。
特异靶向猪MC4R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MC4R基因突变体，可以在构建猪MC4R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MC4R的敲除。
(1)本发明利用质粒载体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)本发明的载体中含有嘌呤霉素抗性基因，利用嘌呤霉素对细胞进行筛选，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MC4R基因的sgRNA导向序列，可通过CRISPR/Cas9 系统敲除或编辑MC4R基因，进而消除MC4R的表达，为制备MC4R转基因猪奠定基础。
附图说明
图1是特异靶向猪MC4R基因的sgRNA导向序列及其位置示意图。
图2是PCR扩增猪MC4R基因外显子片段电泳图。
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MC4R基因的基因组序列(gene ID：NM214173)，设计1个靶向猪MC4R基因的sgRNA。20nt的寡核苷酸sgRNA导向序列为：MC4R-SgRNA1: 5’-TGTGCAGTCCGTAGGTGCTG-3’，位于基因MC4R外显子编码区中；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1 sgRNA导向序列的寡核苷酸序列
核苷酸序列(5’至3’)
Sg1-F CACCTGTGCAGTCCGTAGGTGCTG
Sg1-R AAACCAGCACCTACGGACTGCACA
(2)构建表达sgRNA的载体
质粒载体pX330载体有BbsI酶切位点，用BbsI酶切，其中，酶切体系为10μL体系：BbsI 1μL；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-MC4R-Sg1质粒载体。
实施例2：
(1)猪成纤维细胞的培养与冻存
将猪胎儿置于国产大皿中加生理盐水清洗5-6遍，清洗完毕后，去掉胎儿四肢、尾巴、头和内脏。将处理完的胎儿放于进口小皿中，用剪刀剪碎，剪至最大的组织块为1mm3 即可终止。加2ml 0.25％的EDTA-Trypsin于小皿中，并将小皿放于37度培养箱中消化 25min后用4ml培养液DMEM/F12(10％FBS)终止消化，并用巴斯德吸管吹打并吸出液体放于15ml离心管中，1000rpm，离心3min。离心完后，弃掉上清，加3ml培养液重悬，平均分在三个进口大皿中，进口大皿加8ml培养液DMEM/F12(10％FBS)。第二天观察细胞状态，如细胞量达到90％则按照1:3的比例传代。如细胞量较少，则进行换液即可。一般猪胎儿成纤维细胞传至1代长满后即可冻存，冻存提前配好冻存液放于度中，再进行完常规的消化后用冻存液重悬，500ul分与冻存管中，并迅速将冻存管放在冻盒中，最后将冻存盒放于-80冰箱中过夜第二天，将冻存盒中的细胞导入进口液氮罐中即可。
(2)质粒转染和阳性细胞筛选
将重组质粒pX330-MC4R-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2μg/mL的嘌呤霉素对转染后的细胞进行筛选，筛选持续48h后进行细胞恢复培养，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2所示：
表2 扩增MC4R片段的引物序列
序列(5’至3’)
MC4R-F CTTAAATCAGGTCAGAGGGGATCTC
MC4R-R GGAGAAAGTCTCTTATGCTTGC
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MC4R-CDS。
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MC4R基因的猪成纤维细胞。

Claims (8)

1.一种特异靶向猪MC4R基因的sgRNA导向序列，其特征在于该序列为MC4R-SgRNA1，MC4R-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MC4R基因的sgRNA导向序列在敲除猪MC4R基因中的应用，具体如下：
一、在猪MC4R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转染细胞的方法为脂质体转染法。
5.根据权利要求2所述的应用，其特征在于步骤三中所述筛选所用药物为嘌呤霉素。
6.根据权利要求2所述的应用，其特征在于步骤三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MC4R基因的sgRNA导向序列在特异识别和靶向修饰猪MC4R基因中的应用。
8.权利要求1所述的特异靶向猪MC4R基因的sgRNA导向序列在构建猪MC4R基因突变库中的应用。
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