CN107034218A - 用于猪APN基因编辑的靶向sgRNA、修饰载体及其制备方法和应用 - Google Patents
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Abstract
本发明提供了一种用于猪APN基因编辑的靶向sgRNA、修饰载体及其制备方法和应用,涉及基因工程技术领域,本发明提供的sgRNA和基因修饰载体,特异性强,能够非常高效地通过CRISPR/Cas9n系统在细胞水平上对猪APN基因进行编辑,用获得的APN基因编辑阳性细胞为供体细胞进行体细胞克隆和胚胎移植,得到的APN基因编辑克隆猪具有抗仔猪腹泻病的能力。本发明提供的一种抗仔猪腹泻病的猪的制备方法,破坏了引起仔猪腹泻病的病毒的受体,除了对目的基因APN进行编辑外不会引入其他任何外源基因,也不会对基因组上非APN基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。
Description
技术领域
本发明涉及基因工程技术领域,尤其是涉及一种用于猪APN基因编辑的靶向sgRNA、修饰载体及其制备方法和应用。
背景技术
仔猪腹泻病是初生仔猪和断奶仔猪高发的传染性疾病,给我国养猪业带来巨大的经济损失和危害。目前认为猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)和传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)是引起仔猪腹泻的最主要和最常见的病毒。
许多细胞水平上的研究表明猪氨基肽酶(Porcine aminopeptidase,pAPN)是PEDV和TGEV两种病毒感染猪的主要受体。在生产抗蓝耳病的猪的研究中发现,通过基因编辑技术破坏了蓝耳病病毒受体的猪不会被蓝耳病病毒感染,因此具有抗蓝耳病的能力。所以,对仔猪腹泻病的病毒受体APN的基因进行基因编辑是生产抗仔猪腹泻病的猪的一种重要的可行性方法。
CRISPR/Cas9系统是近年来被广泛应用的一种基因编辑技术,可以精确的对基因组进行定点删除、插入或置换等改造。CRISPR/Cas9系统主要由两个重要的元件构成——导向RNA(sgRNA)和Cas9核酸内切酶(Cas9 nuclease)。很多研究表明,与Cas9核酸内切酶相比,使用突变了一个活性域的Cas9切口酶(Cas9 nickase)和两条sgRNA对目的基因进行编辑可以极大地降低CRISPR/Cas9系统的脱靶效应,这一系统被称之为CRISPR/Cas9n系统。
虽然,目前越来越多的基因作为家畜疾病的生物标志物及治疗靶点被应用,但在针对仔猪腹泻病中的关键基因及其编辑修饰的方法尚未在我国得到应用。
有鉴于此,特提出本发明。
发明内容
本发明的第一个目的在于提供一种用于猪APN基因编辑的靶向sgRNA;
本发明的第二个目的在于提供一种猪APN基因修饰载体;
本发明的第三个目的在于提供一种猪APN基因修饰载体的制备方法;
本发明的第四个目的在于提供一种猪APN基因修饰载体在制备抗仔猪腹泻病的猪中的应用;
本发明的第五个目的在于提供一种抗仔猪腹泻病的猪的制备方法;
本发明的第六个目的在于提供一种抗仔猪腹泻病的猪;以缓解现有技术中存在的针对仔猪腹泻病中的关键基因及其编辑修饰的方法尚未得到应用的技术问题。
本发明提供的一种用于猪APN基因编辑的靶向sgRNA,包括sgRNA-A213和sgRNA-A258;
所述sgRNA-A213的正义链和反义链分别为:
A213-Fwd:5’-CAGGCAACAGCGTTGTGGGT-3’(SEQ ID NO.1);
A213-Rev:5’-ACCCACAACGCTGTTGCCTG-3’(SEQ ID NO.2);
所述sgRNA-A258的正义链和反义链分别为:
A258-Fwd:5’-ACCCTACCTCACTCCCAACG-3’(SEQ ID NO.3);
A258-Rev:5’-CGTTGGGAGTGAGGTAGGGT-3’(SEQ ID NO.4)。
本发明还提供了一种猪APN基因修饰载体,包括上述的两条sgRNA、Cas9切口酶及荧光标记蛋白。
本发明还提供了上述的猪APN基因修饰载体的制备方法,包括以下步骤:
步骤(a):将所述sgRNA-A213的正义链和反义链进行互补配对,形成A213双链DNA分子;
步骤(b):将所述sgRNA-A258的正义链和反义链进行互补配对,形成A258双链DNA分子;
步骤(c):将所述A213双链DNA分子连接到表达载体上,构建表达sgRNA-A213的载体;
步骤(d):将所述A258双链DNA分子连接到表达载体上,构建表达sgRNA-A258的载体;
步骤(e):以所述表达sgRNA-A213的载体为模板,PCR扩增含有U6启动子的所述sgRNA-A213序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-A213序列;
步骤(f):将所述U6-sgRNA-A213序列连接到所述表达sgRNA-A258的载体上,构建完成表达所述sgRNA-A213和sgRNA-A258的载体。
进一步地,在步骤(c)和步骤(d)中,所述表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过BbsⅠ酶切的PX461载体。
进一步地,在步骤(e)中,所述克隆载体为pMD18-T载体。
本发明还提供了上述的猪APN基因修饰载体在制备抗仔猪腹泻病的猪中的应用。
本发明还提供了一种抗仔猪腹泻病的猪的制备方法,应用上述的猪APN基因修饰载体。
进一步地,将所述猪APN基因修饰载体转入猪的体细胞中,获得APN基因编辑阳性细胞克隆,以所述阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得所述抗仔猪腹泻病的猪。
进一步地,所述猪的体细胞为猪成纤维细胞。
另外,本发明还提供了一种抗仔猪腹泻病的猪,应用上述的抗仔猪腹泻病的猪的制备方法制备得到。
本发明提供的用于猪APN基因编辑的靶向sgRNA和包括上述两条sgRNA、Cas9切口酶及荧光标记蛋白的猪APN基因修饰载体,特异性强,能够非常高效地通过CRISPR/Cas9n系统在细胞水平上对猪APN基因进行编辑。本发明提供的抗仔猪腹泻病的猪的制备方法,应用了本发明提供的猪APN基因修饰载体,针对猪仔猪腹泻病中的关键基因APN进行基因编辑,从而破坏PEDV和TGEV感染猪的受体,并且,除了对目的基因APN进行编辑外不会引入其他任何外源基因,也不会对基因组上非APN基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1提供的猪APN基因修饰载体的制备方法的流程图。
具体实施方式
下面将结合附图对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供了一种用于猪APN基因编辑的靶向sgRNA,包括sgRNA-A213和sgRNA-A258;
所述sgRNA-A213的正义链和反义链分别为:
A213-Fwd:5’-CAGGCAACAGCGTTGTGGGT-3’(SEQ ID NO.1);
A213-Rev:5’-ACCCACAACGCTGTTGCCTG-3’(SEQ ID NO.2);
所述sgRNA-A258的正义链和反义链分别为:
A258-Fwd:5’-ACCCTACCTCACTCCCAACG-3’(SEQ ID NO.3);
A258-Rev:5’-CGTTGGGAGTGAGGTAGGGT-3’(SEQ ID NO.4)。
在本发明中,用于猪APN基因编辑的靶向sgRNA能够起到特异性识别猪APN基因的作用。
本发明还提供了一种猪APN基因修饰载体,包括上述的两条sgRNA、Cas9切口酶及荧光标记蛋白,特异性强,能够非常高效地通过CRISPR/Cas9n系统在细胞水平上对猪APN基因进行基因编辑。要生产抗病的猪是需要特异性的对靶基因进行编辑,绝对不能对基因组的其他区域进行编辑,相比于脱靶效应高,在非靶点序列的区域也能进行基因编辑的Cas9系统,本发明选择CRISPR/Cas9n系统而非传统的CRISPR/Cas9系统,利用本发明提供的猪APN基因修饰载体对APN基因进行编辑,旨在在对APN基因进行特异的基因编辑,效率高特异性强的同时,彻底杜绝由于脱靶效应对基因组上其他序列进行基因编辑的可能。
其中,荧光标记蛋白为GFP。
本发明还提供了上述猪APN基因修饰载体的制备方法,包括以下步骤:
步骤(a):将sgRNA-A213的正义链和反义链进行互补配对,形成A213双链DNA分子;
步骤(b):将sgRNA-A258的正义链和反义链进行互补配对,形成A258双链DNA分子;
步骤(c):将A213双链DNA分子连接到表达载体上,构建表达sgRNA-A213的载体;
步骤(d):将A258双链DNA分子连接到表达载体上,构建表达sgRNA-A258的载体;
步骤(e):以表达sgRNA-A213的载体为模板,PCR扩增含有U6启动子的sgRNA-A213序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-A213序列;
步骤(f):将U6-sgRNA-A213序列连接到表达sgRNA-A258的载体上,构建完成表达sgRNA-A213和sgRNA-A258的载体。
其中,在步骤(c)和步骤(d)中,表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过BbsⅠ酶切的PX461载体;表达sgRNA-A213的载体为PX461-A213,表达sgRNA-A258的载体为PX461-A258。
在步骤(e)中,克隆载体为pMD18-T载体,酶切为KpnⅠ酶切。
将连接有sgRNA-A213序列的pMD18-T载体进行KpnⅠ酶切后,获得两端含有KpnⅠ酶切位点的U6-sgRNA-A213序列。
在步骤(f)中,表达sgRNA-A258的载体为经过KpnⅠ酶切的PX461-A258,表达sgRNA-A213和sgRNA-A258的载体为PX461-A258+A213。
将两端含有KpnⅠ酶切位点的U6-sgRNA-A213序列连接到KpnⅠ酶切后的PX461-A258上,构建完成同时表达sgRNA-A213和sgRNA-A258以及Cas9切口酶和GFP的载体PX461-A258+A213。
本发明还提供了上述的猪APN基因修饰载体在制备抗仔猪腹泻病的猪中的应用。
本发明还提供了一种抗仔猪腹泻病的猪的制备方法,应用上述的猪APN基因修饰载体。
在本发明中,将猪APN基因修饰载体转入猪的体细胞,获得APN基因编辑阳性细胞克隆,以阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得抗仔猪腹泻病的猪。
其中,将猪APN基因修饰载体转入猪的体细胞的方法例如可以为,但不限于电穿孔法、显微注射法、磷酸钙共沉淀法或脂质体转染法。
在一个优选的实施方式中,猪的体细胞为猪成纤维细胞,在一个更优选的实施方式中,猪的体细胞为猪胎儿成纤维细胞。
将APN基因编辑后的猪的体细胞作为供体细胞,卵母细胞作为受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入受体猪子宫内妊娠获得APN基因编辑的抗仔猪腹泻病的猪。
本发明提供的抗仔猪腹泻病的猪的制备方法,应用了本发明提供的猪APN基因修饰载体,针对猪仔猪腹泻病中的关键基因APN进行基因编辑,从而破坏猪流行性腹泻病毒和/或传染性胃肠炎病毒受体,并且,除了对目的基因APN进行编辑外不会引入其他任何外源基因,也不会对基因组上非APN基因的区域进行非特异的编辑,遗传背景干净清晰,极大的减少了后期转基因安全评估的工作。
另外,本发明还提供了一种抗仔猪腹泻病的猪,应用上述的抗仔猪腹泻病的猪的制备方法制备得到。
为了有助于更清楚的理解本发明的内容,现结合具体的实施例详细介绍如下。如未明确指出,以下实施例中采用的PX461载体购自Addgene,货号48140;pMD18-T载体购自TaKaRa公司;宿主菌大肠杆菌DH5α购自TaKaRa公司;引物合成由上海生工完成;序列测定由上海睿迪公司完成;质粒小提试剂盒购自TaKaRa公司;LA Taq酶购自TaKaRa公司;T4DNA连接酶购自TaKaRa公司;KpnⅠ内切酶购自TaKaRa公司;BbsⅠ内切酶购自NEB公司;细胞培养基DMEM、PBS、胎牛血清、胰酶和成纤维生长因子(bFGF)购自Life Technologies;脂质体Lipofectamine 2000购自Invitrogen公司;细胞培养板及培养皿购自Thermo Scientific。
实施例1sgRNA的设计和载体的构建
根据NCBI中猪APN基因的DNA序列(如SEQ ID NO.5所示)和mRNA序列(如SEQ IDNO.6所示),在APN基因的编码框区域设计两条sgRNA,为sgRNA-A213和sgRNA-A258,分别在其两端加上粘性接头序列。在每条sgRNA序列F链的5’端加上接头序列CACC,其反向互补序列R链的5’端添加接头序列AAAC,如果sgRNA序列的5’端第一个碱基不是G,那么应先在sgRNA序列F链的5’端添加一个G,再加上接头序列CACC,相应地,其反向互补序列R链的3’端再增加一个C,以便能够与经BbsⅠ酶切的PX461载体的粘性末端互补。
用于构建猪APN基因修饰载体的sgRNA-A213的正义链和反义链分别为:
A213-Fwd:5’-CACCGCAGGCAACAGCGTTGTGGGT-3’(SEQ ID NO.7);
A213-Rev:5’-AAACACCCACAACGCTGTTGCCTGC-3’(SEQ ID NO.8);
所述sgRNA-A258的正义链和反义链分别为:
A258-Fwd:5’-CACCGACCCTACCTCACTCCCAACG-3’(SEQ ID NO.9);
A258-Rev:5’-AAACCGTTGGGAGTGAGGTAGGGTC-3’(SEQ ID NO.10)。
两条sgRNA的正义链和反义链分别在上海生工合成。
将sgRNA-A213和sgRNA-A258的正义链和反义链用水溶解为浓度为200μM的溶液,退火体系如下:
200μM正义链 | 5μL |
200μM反义链 | 5μL |
10×退火缓冲液 | 2μL |
DNase/RNase-free的水 | 8μL |
总体积 | 20μL |
注:10×退火缓冲液的组成包括100mM Tris-HCl(pH8.0),10mM EDTA(pH8.0)和1MNaCl。
在94℃变性5min后,取出样品室温放置10min使sgRNA的正义链和反义链进行互补配对,形成A213双链DNA分子和A258双链DNA分子,-20℃保存。
如图1所示,PX461载体用限制性内切酶BbsⅠ进行酶切回收后,与A213双链DNA分子和A258双链DNA分子连接,连接体系如下:
BbsⅠ线性化的PX461(30ng/μL) | 1μL |
ds oligo(5nM;退火产物1:10000稀释后) | 5μL |
10×T4Ligation Buffer | 2μL |
DNase/RNase-free的水 | 11.9μL |
T4连接酶(350U/μL) | 0.1μL |
总体积 | 20μL |
16℃放置2小时,然后利用常规方法将连接产物转化大肠杆菌DH5α感受态细胞并涂板。待菌落长成后,挑取单菌落扩大培养。将培养好的菌液抽提质粒,测序鉴定sgRNA-A213和sgRNA-A258是否连入载体PX461上。
构建完成同时表达sgRNA-A213、Cas9切口酶以及GFP的载体PX461-A213和同时表达sgRNA-A258、Cas9切口酶以及GFP的载体PX461-A258。
以PX461-A213为模板,sgRNA-Fwd和sgRNA-Rev分别为上下游引物,PCR扩增含有U6启动子的sgRNA-A213序列。
其中,PCR引物序列为:
sgRNA-Fwd:5’-GAGGGCCTATTTCCCATGATTCC-3’(SEQ ID NO.11);
sgRNA-Rev:5’-GGGGTACCTCTAGAGCCATTTG-3’(SEQ ID NO.12)。
PCR的反应体系为:
试剂 | 体积(μL) |
PX461-A213模板(1ng/μL) | 1 |
上游引物sgRNA-Fwd(10μM) | 1 |
下游引物sgRNA-Rev(10μM) | 1 |
dNTP Mix(2.5mM each) | 4 |
10×LA Taq Buffer | 5 |
TaKaRa LA Taq(5U/μL) | 0.25 |
ddH2O | 37.75 |
PCR循环条件如下:
PCR反应完成后,将含有U6启动子的sgRNA-A213序列连接到pMD18-T载体上,然后利用常规方法将连接产物转化大肠杆菌DH5α感受态细胞并涂板。待菌落长成后,挑取单菌落扩大培养。将培养好的菌液抽提质粒,再经过KpnⅠ酶切后获得两端含有KpnⅠ酶切位点的U6-sgRNA-A213序列。
将两端含有KpnⅠ酶切位点的U6-sgRNA-A213序列连接到KpnⅠ酶切后的PX461-A258上,构建完成同时表达sgRNA-A213和sgRNA-A258以及Cas9切口酶和GFP的载体PX461-A258+A213。
实施例2猪的体细胞的转染及APN基因编辑细胞克隆点的筛选
细胞培养及瞬时转染:
接种猪胎儿成纤维细胞于6孔细胞培养板中,在含有15%胎牛血清的DMEM培养基中培养至50-70%汇合度时,按照说明书要求,用Lipofectamine 2000脂质体将实施例1提供的载体PX461-A258+A213转入细胞中。
转染后细胞的流式分选及单克隆培养:
转染后的细胞在37℃培养箱中培养48小时,用0.1%的胰酶消化后,流式细胞仪分选表达GFP的阳性细胞,按照50-100个细胞/100mm培养皿的密度接种细胞,在含有15%胎牛血清及2.5ng/mL成纤维细胞生长因子(bFGF)的DMEM培养基中培养9至12天,至长出明显的单细胞克隆点。
单细胞克隆点APN基因编辑情况的鉴定:
将生长状态良好的单细胞克隆点细胞用0.1%的胰酶消化后转接到48孔细胞培养板中,待细胞长满需要传代时,取1/10的细胞裂解液为模板做细胞PCR,剩余细胞接种到24孔细胞培养板中继续培养至长满然后冻存到液氮中保存。
用来鉴定细胞克隆点APN基因编辑情况的引物为:
引物名称 | 引物序列(5’-3’) | SEQ ID NO. |
APN797F | GGGATATAAGCCTGGTCCGAAG | 13 |
APN1416R | AAGTTCCCCCTGGAATTCACTC | 14 |
其中,PCR的反应体系为:
PCR的循环条件为:
随机挑取19个细胞克隆点的PCR产物,胶回收后连接到pMD18-T载体上,测序分析后发现其中16个细胞克隆点发生了APN基因的编辑,为阳性细胞,基因编辑效率高达84.21%。因此,本发明提供的同时表达两条sgRNA和Cas9切口酶的猪APN基因修饰载体,可以非常高效地对靶基因APN进行基因编辑。发生了基因编辑的细胞克隆点基因型结果如下表所示。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 浙江大学
<120> 用于猪APN基因编辑的靶向sgRNA、修饰载体及其制备方法和应用
<160> 14
<170> PatentIn version 3.5
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caggcaacag cgttgtgggt 20
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acccacaacg ctgttgcctg 20
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accctacctc actcccaacg 20
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ggattttgag gttctactat atggtgttta atatgttttc taacattaaa tccgctcacc 60
aaatctgaga cgtaaattct agtatttatt tatgtgaaca gggttctcag aaaggagaac 120
ttacctgcca gaggtcatgg ctgggaagag gttaagccgc cgctagcctc ccttctttaa 180
aaaaaaaaaa aaaaaaaaaa aaaggcaaaa caacttattt cattctactc agtgagctga 240
taattgaggg gaaagttttt ggcaagaagg gaaagtggcg gggggaggac ctggaagaac 300
tccctgctct ggaagaatgc gggaggctgg gaccatgtcc ctgaggagcg ccgggcatcc 360
ctccaactgc agggctgacc cggtgtggtc ttgacccgag ccagaggccg gctctccccg 420
tcttttcacc tcccacctct tgctcctggg acgtccttcg accctcctgg atctaacctc 480
agtcttcctg ctcctgtgcc tgttgtcata gctcacagct cacagggaga tccaagccac 540
ctggccgctc cctctccccg ctgggccagc tgcctgccac ctgcccttca gcccttggtg 600
ggctcccagg ctcctgcagc ctgtaaccag accctgtttg ctcccagcag gcacccctga 660
gccgcactcc gcacgctgtt cctgaatctc ccctccagaa ccggagcagt gtctctaccc 720
agttcagtga ccttcgtctg tctgagccct ggttaatttt tgcccagtct gcaggctgtg 780
gggctcctcc ccttcaggga tataagcctg gtccgaagct gccctgtccc ctgcccgtcc 840
tgagcctccc cgagctccct tctcaccctc accatggcca agggattcta catttccaag 900
gccctgggca tcctgggcat cctcctcggc gtggcggccg tggccaccat catcgctctg 960
tctgtggtgt acgcccagga gaagaacaag aatgccgagc atgtccccca ggcccccacg 1020
tcgcccacca tcaccaccac agccgccatc accttggacc agagcaagcc gtggaaccgg 1080
taccgcctac ccacaacgct gttgcctgat tcctacaacg tgacgctgag accctacctc 1140
actcccaacg cggatggcct gtacatcttc aagggcaaaa gcatcgtccg cttcatctgc 1200
caggagccca ccgatgtcat catcatccat agcaagaagc tcaactacac cacccagggg 1260
cacatggtgg tcctgcgggg cgtgggggac tcccaggtcc cagagatcga caggactgag 1320
ctggtagagc tcactgagta cctggtggtc cacctcaagg gctcgctgca gcccggccac 1380
atgtacgaga tggagagtga attccagggg gaacttgccg acgacctggc aggcttctac 1440
cgcagcgagt acatggaggg caacgtcaaa aagtaagtca ggtgggggca caccctagat 1500
gctgaggcag agctggatcc tgggggccaa ggaagggctt ggattcggga ccttggaacc 1560
ttctggagac tttggctggc ccgtcgctcc atccgcagct ctggtagaga agctatctag 1620
acaatcagcc ctttcccgga gagcccccct aaccttaggg agtcaggggt gagtgatcca 1680
agtgccccct tgggtagaaa ggaaaacagg ctctgaggac agaaatttgc ccaaggtctc 1740
ccagctaatt caggggtgga gcctgcccgg actttgaccc caagtccaga aggagctctg 1800
ctctcccaag tcagctggcc tgtcagcctg gaggcggcct gggggaggcg gggagggcag 1860
ggatggggct gtgcacccct ttccatgccc agccagccat ggcctacacc ccccaccccc 1920
ggccaccccc atgggcacag gcattttgct ggcatacctt ctaaccccct gcttcgggca 1980
gggtgctggc cacgacacag atgcagtcta cagatgcccg gaaatccttc ccatgctttg 2040
acgagccagc catgaaggcc acgttcaaca tcactctcat ccaccctaac aacctcacgg 2100
ccctgtccaa tatgccgccc aaaggtgagc gggcctggcg gggaccacac ggcctgggaa 2160
agcaggtccc tggggctggg gtgcaggtcc ctgttgctgg ggtgcaggcc caggaagagg 2220
gcacccctcc acgcctgcgt gtcgcaccca ggttccagca ccccacttgc agaagacccc 2280
aactggtctg tcactgagtt cgaaaccaca cctgtgatgt ccacgtacct tctggcctac 2340
atcgtgagcg agttccagag cgtgaatgaa acggcccaaa atggcgtcct ggtaaggggc 2400
tgagcccacc tgcccttccc cacattggcc ctggcctggg aagtattccc atttatcctc 2460
atccttgtcc ctgtgcttga gtcgtgaggc agtgtttgaa ttccagctct gagtcatctt 2520
gggcaaatgt cccaagttct ctgaccttca gtctctgcat ctgaaaaatg ggaccctcct 2580
catgaaggga gttcctggcc cctgaatgcc agacagatag cagctgagtc tgtggttatt 2640
ccccaaaggc tcaaagctcc gcagggacac cccctttacc gccccaccgc ccccgccacc 2700
ctcttctctg ctgaccaaac ctccacttta acctggtttg tccccctgac tctgggactt 2760
ggcccaccag caccaggacc caaggggggc cctgacccac ctctatcttt gcagatccgg 2820
atctgggctc ggcctaatgc aattgcagag ggccatggca tgtatgccct gaatgtgaca 2880
ggtcccatcc taaacttctt tgccaatcat tataatacac cctacccact ccccaaatcc 2940
g 2941
<210> 6
<211> 3387
<212> RNA
<213> Sus scrofa(猪)
<400> 6
ccctgcccgt cctgagcctc cccgagctcc cttctcaccc tcaccatggc caagggattc 60
tacatttcca aggccctggg catcctgggc atcctcctcg gcgtggcggc cgtggccacc 120
atcatcgctc tgtctgtggt gtacgcccag gagaagaaca agaatgccga gcatgtcccc 180
caggccccca cgtcgcccac catcaccacc acagccgcca tcaccttgga ccagagcaag 240
ccgtggaacc ggtaccgcct acccacaacg ctgttgcctg attcctactt cgtgacgctg 300
agaccctacc tcactcccaa cgcggatggc ctgtacatct tcaagggcaa aagcatcgtc 360
cgcttactct gccaggagcc caccgatgtc atcatcatcc atagcaagaa gctcaactac 420
accacccagg ggcacatggt ggtcctgcgg ggcgtggggg actcccaggt cccagagatc 480
gacaggactg agctggtaga gctcactgag tacctggtgg tccacctcaa gggctcgctg 540
cagcccggcc acatgtacga gatggagagt gaattccagg gggaacttgc cgacgacctg 600
gcaggcttct accgcagcga gtacatggag ggcaacgtca aaaaggtgct ggccacgaca 660
cagatgcagt ctacagatgc ccggaaatcc ttcccatgct ttgacgagcc agccatgaag 720
gccacgttca acatcactct catccaccct aacaacctca cggccctgtc caatatgccg 780
cccaaaggtt ccagcacccc acttgcagaa gaccccaact ggtctgtcac tgagttcgaa 840
accacacctg tgatgtccac gtaccttctg gcctacatcg tgagcgagtt ccagagcgtg 900
aatgaaacgg cccaaaatgg cgtcctgatc cggatctggg ctcggcctaa tgcaattgca 960
gagggccatg gcatgtatgc cctgaatgtg acaggtccca tcctaaactt ctttgccaat 1020
cattataata catcctaccc actccccaaa tccgaccaga ttgccttgcc cgacttcaat 1080
gccggtgcca tggagaactg ggggctggtg acctaccggg agaacgcgct gctgtttgac 1140
ccacagtcct cctccatcag caacaaagag cgagttgtca ctgtgattgc tcacgagctg 1200
gcccaccagt ggtttggcaa cctggtgacc ctggcctggt ggaatgacct gtggctgaat 1260
gagggctttg cctcctatgt ggagtacctg ggtgctgacc acgcagagcc cacctggaat 1320
ctgaaagacc tcatcgtgcc aggcgacgtg taccgagtga tggctgtgga tgctctggct 1380
tcctcccacc cgctgaccac ccctgctgag gaggtcaaca cacctgccca gatcagcgag 1440
atgtttgact ccatctccta cagcaaggga gcctcggtta tcaggatgct ctccaacttc 1500
ctgactgagg acctgttcaa ggagggcctg gcgtcctact tgcatgcctt tgcctatcag 1560
aacaccacct acctggacct gtgggagcac ctgcagaagg ctgtggatgc tcagacgtcc 1620
atcaggctgc cagacactgt gagagccatc atggatcgat ggaccctgca gatgggcttc 1680
cccgtcatca ccgtggacac caagacagga aacatctcac agaagcactt cctcctcgac 1740
tccgaatcca acgtcacccg ctcctcagcg ttcgactacc tctggattgt tcccatctca 1800
tctattaaaa atggtgtgat gcaggatcac tactggctgc gggatgtttc ccaagcccag 1860
aatgatttgt tcaaaaccgc atcggacgat tgggtcttgc tgaacgtcaa cgtgacaggc 1920
tatttccagg tgaactacga cgaggacaac tggaggatga ttcagcatca gctgcagaca 1980
aacctgtcgg tcatccctgt catcaatcgg gctcaggtca tctacgacag cttcaacctg 2040
gccactgccc acatggtccc tgtcaccctg gctctggaca acaccctctt cctgaacgga 2100
gagaaagagt acatgccctg gcaggccgcc ctgagcagcc tgagctactt cagcctcatg 2160
ttcgaccgct ccgaggtcta tggccccatg aagaaatacc tcaggaagca ggtcgaaccc 2220
ctcttccaac atttcgaaac tctcactaaa aactggaccg agcgcccaga aaatctgatg 2280
gaccagtaca gtgagattaa tgccatcagc actgcctgct ccaatggatt gcctcaatgt 2340
gagaatctgg ccaagaccct tttcgaccag tggatgagcg acccagaaaa taacccgatc 2400
caccccaacc tgcggtccac catctactgc aatgccatag cccagggcgg ccaggaccag 2460
tgggactttg cctgggggca gttacaacaa gcccagctgg taaatgaggc cgacaaactc 2520
cgctcagcgc tggcctgcag caacgaggtc tggctcctga acaggtacct gggttacacc 2580
ctgaacccgg acctcattcg gaagcaagac gccacctcca ctattaacag cattgccagc 2640
aatgtcatcg ggcagcctct ggcctgggat tttgtccaga gcaactggaa gaagctcttt 2700
caggactatg gcggtggttc cttctccttc tccaacctca tccagggtgt gacccgaaga 2760
ttctcctctg agtttgagct gcagcagctg gagcagttca agaagaacaa catggatgtg 2820
ggcttcggct ccggcacccg ggctctggag caagccctgg agaagaccaa ggccaacatc 2880
aagtgggtga aggagaacaa ggaggtggtg ttgaattggt tcatagagca cagctaatag 2940
tgcctggtcc ttcccgccac ctggcccccc gcacaagatg cccgcatgtg tccatcccag 3000
ggcccacggc agggcccatg ttcctgaagc ccgaggcacc tgcgtcctcc ctttagggac 3060
aaagcctgtg gcccatgtta tctccattct gccctggggg ccaatccagt ttctggtgac 3120
cagactgtcc aggtgtctcc cagccactgc cccttgtgcc aaccccaccc tgggcctggc 3180
ccagggccct tctcagggaa gtccagctcc agggccagat gagcagaagc ccttgatgga 3240
tgatggatgg ccttgaagaa ctgccctcta ccctctctcc cccttttcca taaagaccct 3300
gaacctgaga atcaacaggg catcagatct gtatattttt tttctaggag taaatgtaaa 3360
taaaggattt ctagatgaaa ggaattc 3387
<210> 7
<211> 25
<212> DNA
<213> 人工序列
<400> 7
caccgcaggc aacagcgttg tgggt 25
<210> 8
<211> 25
<212> DNA
<213> 人工序列
<400> 8
aaacacccac aacgctgttg cctgc 25
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
caccgaccct acctcactcc caacg 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
aaaccgttgg gagtgaggta gggtc 25
<210> 11
<211> 23
<212> DNA
<213> 人工序列
<400> 11
gagggcctat ttcccatgat tcc 23
<210> 12
<211> 22
<212> DNA
<213> 人工序列
<400> 12
ggggtacctc tagagccatt tg 22
<210> 13
<211> 22
<212> DNA
<213> 人工序列
<400> 13
gggatataag cctggtccga ag 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列
<400> 14
aagttccccc tggaattcac tc 22
Claims (10)
1.一种用于猪APN基因编辑的靶向sgRNA,其特征在于,所述sgRNA包括sgRNA-A213和sgRNA-A258;
所述sgRNA-A213的正义链和反义链分别为:
A213-Fwd:5’-CAGGCAACAGCGTTGTGGGT-3’(SEQ ID NO.1);
A213-Rev:5’-ACCCACAACGCTGTTGCCTG-3’(SEQ ID NO.2);
所述sgRNA-A258的正义链和反义链分别为:
A258-Fwd:5’-ACCCTACCTCACTCCCAACG-3’(SEQ ID NO.3);
A258-Rev:5’-CGTTGGGAGTGAGGTAGGGT-3’(SEQ ID NO.4)。
2.一种猪APN基因修饰载体,其特征在于,包括权利要求1所述的两条sgRNA、Cas9切口酶及荧光标记蛋白。
3.如权利要求2所述的猪APN基因修饰载体的制备方法,其特征在于,包括以下步骤:
步骤(a):将所述sgRNA-A213的正义链和反义链进行互补配对,形成A213双链DNA分子;
步骤(b):将所述sgRNA-A258的正义链和反义链进行互补配对,形成A258双链DNA分子;
步骤(c):将所述A213双链DNA分子连接到表达载体上,构建表达sgRNA-A213的载体;
步骤(d):将所述A258双链DNA分子连接到表达载体上,构建表达sgRNA-A258的载体;
步骤(e):以所述表达sgRNA-A213的载体为模板,PCR扩增含有U6启动子的所述sgRNA-A213序列,并连接到克隆载体上,再经过酶切后获得U6-sgRNA-A213序列;
步骤(f):将所述U6-sgRNA-A213序列连接到所述表达sgRNA-A258的载体上,构建完成表达所述sgRNA-A213和sgRNA-A258的载体。
4.根据权利要求3所述的制备方法,其特征在于,在步骤(c)和步骤(d)中,所述表达载体为带有Cas9切口酶、荧光标记蛋白及U6启动子的经过Bbs Ⅰ酶切的PX461载体。
5.根据权利要求3所述的制备方法,其特征在于,在步骤(e)中,所述克隆载体为pMD18-T载体。
6.如权利要求2所述的猪APN基因修饰载体在制备抗仔猪腹泻病的猪中的应用。
7.一种抗仔猪腹泻病的猪的制备方法,其特征在于,应用权利要求2所述的猪APN基因修饰载体。
8.根据权利要求7所述的抗仔猪腹泻病的猪的制备方法,其特征在于,将所述猪APN基因修饰载体转入猪的体细胞中,获得APN基因编辑阳性细胞克隆,以所述阳性细胞为供体细胞进行体细胞克隆和胚胎移植,获得所述抗仔猪腹泻病的猪。
9.根据权利要求8所述的抗仔猪腹泻病的猪的制备方法,其特征在于,所述猪的体细胞为猪成纤维细胞。
10.一种抗仔猪腹泻病的猪,其特征在于,应用权利要求7-9任一项所述的抗仔猪腹泻病的猪的制备方法制备得到。
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