CN112011539A - 一种基于CRISPR-Cas9技术靶向敲除猪GDPD2基因的细胞系及其构建方法 - Google Patents
一种基于CRISPR-Cas9技术靶向敲除猪GDPD2基因的细胞系及其构建方法 Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR‑Cas9技术靶向敲除猪GDPD2基因的细胞系及其构建方法,该细胞系采用以下步骤制备:(1)根据猪GDPD2基因设计sgRNA向导序列;(2)将正链sgRNA序列和负链sgRNA序列退火形成双链DNA;将双链DNA与线性化的pGK1.1载体连接获得阳性打靶载体;(3)将阳性打靶载体混合电转染靶细胞IPEC‑J2获得GDPD2基因敲除的IPEC‑J2细胞。本发明首次利用CRISPR/Cas9技术建立猪GDPD2基因敲除的IPEC‑J2细胞系,有助于研究GDPD2的蛋白功能。CRISPR/Cas9敲除技术方法简单,设计sgRNA即可对靶基因进行高效敲除,导致GDPD2基因功能的缺失,是理想的GDPD2基因敲除的IPEC‑J2细胞模型。
Description
技术领域
本发明属于基因工程技术领域,涉及利用CRISPR/Cas9技术构建猪GDPD2基因敲除细胞系,也涉及猪小肠上皮细胞系(IPEC-J2)基因敲除模型的构建技术。
背景技术
CRISPR/Cas9系统是古细菌和细菌在长期发展过程中形成的适应性免疫防御系统。作为规律成簇间隔短回文重复系统,CRISPR/Cas9是一种基因编辑工具,通过Cas9核酸酶介导sgRNA识别并切割dsDNA,诱发由非同源末端连接修复机制造成的移码突变,从而实现对靶基因的编辑。由于该技术操作性强且效率高,近几年已经成为定点编辑的重要遗传手段,是最具有临床与应用前景的基因治疗技术之一。
猪小肠上皮细胞系(IPEC-J2)是来源于仔猪空肠中的柱状上皮细胞,是研究猪多种疾病的理想体外模型,广泛应用于产肠毒素大肠杆菌和病毒性腹泻抗性基因的功能鉴定。
GDPD2是甘油磷酸二酯磷酸二酯酶(GDPD)家族成员,是一个蛋白质编码基因,这种蛋白在成骨细胞分化和生长中起调控作用,并参与细胞的形态变化。目前尚未有猪GDPD2基因的敲除载体及GDPD2基因敲除的小肠上皮细胞模型相关研究报道。
发明内容
发明的目的在于提供一组基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的sgRNA向导序列。
本发明的另一目的在于提供一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的双链DNA。
本发明的另一目的在于提供一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的敲除载体及其在敲除猪小肠上皮细胞系(IPEC-J2)中GDPD2基因的应用。
本发明的又一目的在于提供一种基于CRISPR/Cas9技术特异性靶向敲除猪GDPD2基因的细胞系。
本发明的又一目的在于提供一种基于CRISPR/Cas9技术靶向敲除猪小肠上皮细胞系(IPEC-J2)中GDPD2基因的方法。
本发明的目的可以通过以下技术方案实现:
一组基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的sgRNA向导序列,该sgRNA向导序列包括sgRNA1、sgRNA2和sgRNA3:
sgRNA1:CTCACCATGGCCGAGTCCCGCGG;
sgRNA2:CCAACAAGGTGAAGTATGGGTGG;
sgRNA3:ACTGTCTGTATAGCTGCCACTGG。
一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的双链DNA,将上述sgRNA向导序列的5’端添加CACC并去除3’端的PAM(NGG)序列,形成正链sgRNA序列(需要注意的是,如果sgRNA 5’端的第一个碱基不是G,则添加CACCG),将sgRNA向导序列的反向互补序列的5’端添加碱基AAAC,形成负链sgRNA序列;将正链sgRNA序列和负链sgRNA序列退火形成双链DNA,该双链DNA包括(1)~(3)组双链DNA:
(1)GDPD2-1F:CACCGCTCACCATGGCCGAGTCCCG,GDPD2-1R:AAACCGGGACTCGGCCATGGTGAGC;
(2)GDPD2-2F:CACCGCCAACAAGGTGAAGTATGGG,GDPD2-2R:AAACCCCATACTTCACCTTGTTGGC;
(3)GDPD2-3F:CACCGACTGTCTGTATAGCTGCCAC,GDPD2-3R:AAACGTGGCAGCTATACAGACAGTC。
一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的敲除载体,该载体采用以下步骤构建:
(1)靶位点设计及sgRNA序列的合成:依据猪GDPD2基因CDS区所在的第一个外显子设计如上述的sgRNA向导序列,将各sgRNA向导序列的5’端添加CACC并去除3’端的PAM(NGG)序列,若sgRNA 5’端第一个碱基不是G,则添加CACCG,形成正链sgRNA序列,将各sgRNA向导序列的反向互补序列的5’端添加碱基AAAC,形成负链sgRNA序列;
(2)载体构建:将所述的正链sgRNA序列和负链sgRNA序列退火形成如上述的三组双链DNA;将各组双链DNA分别与线性化的pGK1.1载体连接,得到连接产物并进行筛选获得三种阳性打靶载体即为靶向敲除猪GDPD2基因的敲除载体。
一种基于CRISPR/Cas9技术特异性靶向敲除猪GDPD2基因的细胞系,该细胞系为将上述的三种阳性打靶载体混合电转入IPEC-J2细胞并进行筛选和验证得到的;三种阳性打靶载体可以等比例混合电转,但不限于此。
一种基于CRISPR/Cas9技术靶向敲除猪小肠上皮细胞系中GDPD2基因的方法,该方法包括以下步骤:
(1)靶位点设计及sgRNA序列的合成:依据猪GDPD2基因CDS区所在的第一个外显子设计如上述的sgRNA向导序列,将各sgRNA向导序列的5’端添加CACC并去除3’端的PAM序列,若sgRNA 5’端第一个碱基不是G,则添加CACCG,形成正链sgRNA序列,将各sgRNA向导序列的反向互补序列的5’端添加碱基AAAC,形成负链sgRNA序列;
(2)载体构建:将所述的正链sgRNA序列和负链sgRNA序列退火形成如权利要求2中所述的三组双链DNA;将各组双链DNA分别与线性化的pGK1.1载体连接,得到连接产物并进行筛选获得三种阳性打靶载体即为靶向敲除猪GDPD2基因的敲除载体;
(3)细胞转染:将三种阳性打靶载体混合电转染靶细胞IPEC-J2并进行筛选验证和培养获得猪GDPD2基因靶向敲除的IPEC-J2细胞。
作为一种优选技术方案,上述方法的步骤(2)中得到连接产物并进行筛选获得三种阳性打靶载体的过程为:将连接产物转化大肠杆菌,挑取单克隆,摇菌培养后,根据sgRNA向导序列设计菌液PCR引物组,进行PCR筛选验证获得三种阳性打靶载体;所述菌液PCR引物组包含三对引物对,各引物对的正向引物为通用引物,反向引物分别为sgRNA反向互补序列,各引物对的具体序列如下所示:
F1:CATATGCTTACCGTAACTTGAAAG,
R1:CGGGACTCGGCCATGGTGAG;
F2:CATATGCTTACCGTAACTTGAAAG,
R2:CCCATACTTCACCTTGTTGG;
F3:CATATGCTTACCGTAACTTGAAAG,
R3:GTGGCAGCTATACAGACAGT。
作为一种优选技术方案,上述方法的步骤(3)中所述筛选验证的过程为:
(Ⅰ)将三种阳性打靶载体混合电转染靶细胞IPEC-J2,经嘌呤霉素药筛,提取细胞基因组DNA,在敲除靶位点附近设计高特异性的引物进行PCR扩增,引物序列为:GDPD2-seqF:CCCCGGGCTCCTCAAATATC;GDPD2-seqR:TTCCTCCTGGGGAGAGAAAG;
(Ⅱ)CruiserTM酶切PCR扩增产物鉴定有效打靶的阳性克隆;
(Ⅲ)对CruiserTM酶切筛选的GDPD2基因敲除细胞进行TA克隆测序,获得GDPD2基因敲除的IPEC-J2细胞。
上述的sgRNA向导序列、双链DNA、敲除载体或敲除猪GDPD2基因的细胞系在猪抗病育种中的应用。
上述的方法在猪抗病育种中的应用。
本发明技术方案的详细步骤为:
(1)通过在线软件(http://crispr.mit.edu/)设计sgRNA向导序列,包括sgRNA1、sgRNA2、sgRNA3,序列如下:
sgRNA1:CTCACCATGGCCGAGTCCCGCGG;
sgRNA2:CCAACAAGGTGAAGTATGGGTGG;
sgRNA3:ACTGTCTGTATAGCTGCCACTGG。
(2)在5’端添加额外碱基CACCG并去除3’端的PAM序列,反向互补序列添加额外碱基AAAC,退火后形成dsDNA。
(3)退火后dsDNA分别与线性化敲除载体PGK1.1连接,得到连接产物。后续进行菌液PCR的筛选并扩大培养,提取质粒得到三种阳性打靶载体。
(4)三种阳性打靶载体混合电转入IPEC-J2细胞系并进行药筛,提取GDPD2敲除细胞DNA,并进行PCR的扩增。
(5)CruiserTM酶切PCR产物以及PCR产物的测序验证,建立了GDPD2基因敲除细胞系。
本发明采用CRISPR/Cas9技术敲除IPEC-J2细胞系中GDPD2的基因序列,建立GDPD2基因的敲除细胞系,为进一步验证GDPD2基因在猪抗病育种中的功能,揭示GDPD2基因的作用机制。
本发明利用CRISPR-Cas9特异性靶向敲除猪GDPD2基因的技术中,sgRNA向导序列的设计起关键作用,sgRNA设计不好,后面单克隆筛选的细胞即没有敲除效率,因此sgRNA的序列为本发明的重要发明点之一。本发明通过实验从众多的sgRNA可能的序列中筛选出可以高效敲除猪GDPD2基因sgRNA向导序列。
本发明的有益效果:
(1)首次利用CRISPR/Cas9技术建立猪GDPD2基因敲除的IPEC-J2细胞系。
(2)CRISPR/Cas9敲除技术比起干扰、基因沉默等技术手段更有效果,能够有助于我们研究GDPD2的蛋白功能。
(3)CRISPR/Cas9敲除技术方法简单,设计sgRNA即可对靶基因进行高效敲除,且敲除测序结果显示GDPD2基因序列缺失,导致GDPD2基因功能的缺失,是比较理想的GDPD2基因敲除的IPEC-J2细胞模型。
附图说明
图1:菌液PCR凝胶电泳检测图;其中,M为100bp DNAMarker,1-6均为阳性菌液PCR条带,条带大小均为100bp。
图2:GDPD2基因敲除载体测序峰图;其中,A为sgRNA1对应的测序峰图,B为sgRNA2对应的测序峰图,C为sgRNA3对应的测序峰图。
图3:pool细胞测序峰图初步验证GDPD2基因的敲除效率。
图4:CruiserTM酶切筛选GDPD2基因敲除细胞DNA PCR产物凝胶电泳检测图。
图5:GDPD2基因敲除细胞的DNA序列与未敲除细胞DNA序列比对分析结果。
具体实施方式
以下结合具体实施例对本发明做出详细的描述。根据以下的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明做出各种改变和修改,以使其适用各种用途和条件。
实施例1
1.靶位点得设计及sgRNA序列的合成
根据NCBI数据库(https://www.ncbi.nlm.nih.gov/)找到GDPD2(Gene ID:100516309)CDS区,依据CDS区所在的第一个外显子进行敲除靶位点的设计,运用CRISPRDesign(http://crispr.mit.edu/)设计三条sgRNA向导序列:
sgRNA1:CTCACCATGGCCGAGTCCCGCGG
sgRNA2:CCAACAAGGTGAAGTATGGGTGG
sgRNA3:ACTGTCTGTATAGCTGCCACTGG
sgRNA向导序列5’端添加CACC并去除3’端的PAM(NGG)序列,若sgRNA向导序列5’端第一个碱基不是G,则添加CACCG,形成正链sgRNA序列;sgRNA向导序列的反向互补序列5’端添加碱基AAAC形成负链sgRNA序列。三对正负链sgRNA序列如下:
GDPD2-1F:CACCGCTCACCATGGCCGAGTCCCG
GDPD2-1R:AAACCGGGACTCGGCCATGGTGAGC;
GDPD2-2F:CACCGCCAACAAGGTGAAGTATGGG
GDPD2-2R:AAACCCCATACTTCACCTTGTTGGC;
GDPD2-3F:CACCGACTGTCTGTATAGCTGCCAC
GDPD2-3R:AAACGTGGCAGCTATACAGACAGTC。
2.GDPD2基因敲除载体的构建
(1)将上述合成的三对正负链sgRNA序列分别进行退火,获得双链sgRNA序列,退火反应体系如下:
PCR反应程序:37℃30min,95℃5min,梯度降温至5℃,自然冷却20min。
(2)BbsI酶对PGK1.1载体(购自Genloci,货号GP0132)进行线性化处理,酶切反应体系如下:
酶切体系37℃孵育3h,后对线性化载体进行切胶回收。
(3)线性化载体PGK1.1与各组双链sgRNA序列分别连接,连接反应体系如下:
上述连接体系16℃连接过夜,获得连接产物。
(4)取Top10感受态置于冰上融解,加入10μL连接产物,混匀,冰上静置30min,42℃水浴热激60s,迅速拿出置于冰上静置2-3min。向管中加入无抗SOC液体培养基800μL,摇床(37℃/160rpm)孵育45min,4500rpm离心5min,弃去管中800μL上清,沉淀吹散混匀在100μL上清中,均匀涂布在提前准备好的含卡那霉素抗性的固体培养基平板上,37℃倒置培养12-16h。
设计菌液PCR引物,正向引物为通用引物,反向引物分别为sgRNA反向互补序列:
F1:CATATGCTTACCGTAACTTGAAAG
R1:CGGGACTCGGCCATGGTGAG;
F2:CATATGCTTACCGTAACTTGAAAG
R2:CCCATACTTCACCTTGTTGG;
F3:CATATGCTTACCGTAACTTGAAAG
R3:GTGGCAGCTATACAGACAGT。
菌液PCR筛选:从培养板上挑选单菌落加入含1mL卡那霉素抗性的LB培养基,摇床摇7-8h。PCR管中加入上述正反向引物各1μL,菌液8μL,普通Mix10μL。反应程序:95℃10min,95℃30s,60℃30s,72℃30s,35-40个循环,72℃10min。
图1为菌液PCR凝胶电泳检测图,M为100bp DNA Marker,1-6均为阳性菌液PCR条带,条带大小均为100bp,说明获得了阳性打靶载体。
对得到的阳性菌落进行进一步测序验证,测序结果如图2所示,从测序图中可以找出sgRNA序列,表明sgRNA序列已与PGK1.1载体成功连接,获得GDPD2敲除载体。对阳性菌落进行扩大培养,提取GDPD2敲除载体,共获得三种阳性打靶载体用于后续实验。
3.细胞转染
将状态良好处于对数生长期的约为5×106个猪小肠上皮细胞(IPEC-J2)置于15mL离心管中,1000rpm离心4min,弃上清,后将细胞沉淀悬浮于210μL DPBS中,转移至1.5mL离心管,取上述三种阳性打靶载体等比例混合加到离心管中,混匀。使用电转法进行转染,之后将细胞移至六孔板培养基中培养。
4.GDPD2基因的敲除
培养72h后以终浓度3μg/mL的嘌呤霉素进行药筛,待细胞长满之后进行pool细胞测序,结果如图3所示,测序结果显示有套峰,初步证明敲除有效率。
用有限稀释法稀释药筛后细胞到10块96孔板中,37℃CO2培养箱中培养,定期观察单克隆的生长情况,之后转移至48孔板中扩大培养,当细胞长满48孔板时,分离出部分细胞并用DNA提取试剂盒提取细胞基因组DNA。
5.GDPD2基因敲除细胞的筛选与验证
(1)PCR扩增目的片段:在GDPD2敲除靶位点附近设计高特异性引物,扩增产物长度为333bp,引物序列如下:
GDPD2-seqF:CCCCGGGCTCCTCAAATATC;
GDPD2-seqR:TTCCTCCTGGGGAGAGAAAG。
PCR扩增体系反应如下:
反应程序为:95℃5min,95℃30s,55-62℃30s,72℃30s,35个循环,72℃10min。
(2)CruiserTM酶切筛选GDPD2敲除细胞:使用CruiserTM酶对上述PCR扩增产物进行酶切,酶切反应体系如下:
37℃孵育3h后进行琼脂糖凝胶电泳检测。
图4为CruiserTM酶切GDPD2基因敲除细胞结果,产物扩增长度333bp,酶切之后片段长度明显变小的克隆为阳性克隆,图中82#克隆初步鉴定为有效打靶的阳性克隆。
(3)对CruiserTM酶切筛选出来的GDPD2基因敲除细胞做TA克隆测序,测序结果与野生型细胞(未进行基因敲除的IPEC-J2细胞)DNA序列比对,结果如图5,GDPD2基因敲除的细胞发生52bp的碱基缺失。
通过与未敲除细胞的DNA序列对比,可以看出本发明采用以上方法成功构建了GDPD2基因敲除的猪小肠上皮细胞系(IPEC-J2),缺失片段为52bp,缺失序列如下:CCCGCGGCTGCTGCTCTGTCTGGGCCCGCTGCCTCCACTGTCTGTATAGCTG。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 扬州大学
<120> 一种基于CRISPR-Cas9技术靶向敲除猪GDPD2基因的细胞系及其构建方法
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Claims (9)
1.一组基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的sgRNA向导序列,其特征在于,该sgRNA向导序列包括sgRNA1、sgRNA2和sgRNA3:
sgRNA1:CTCACCATGGCCGAGTCCCGCGG;
sgRNA2:CCAACAAGGTGAAGTATGGGTGG;
sgRNA3:ACTGTCTGTATAGCTGCCACTGG。
2.一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的双链DNA,其特征在于,该双链DNA包括(1)~(3)组双链DNA:
(1)GDPD2-1F:CACCGCTCACCATGGCCGAGTCCCG,GDPD2-1R:AAACCGGGACTCGGCCATGGTGAGC;
(2)GDPD2-2F:CACCGCCAACAAGGTGAAGTATGGG,GDPD2-2R:AAACCCCATACTTCACCTTGTTGGC;
(3)GDPD2-3F:CACCGACTGTCTGTATAGCTGCCAC,GDPD2-3R:AAACGTGGCAGCTATACAGACAGTC。
3.一种基于CRISPR/Cas9技术靶向敲除猪GDPD2基因的敲除载体,其特征在于,该载体采用以下步骤构建:
(1)靶位点设计及sgRNA序列的合成:依据猪GDPD2基因CDS区所在的第一个外显子设计如权利要求1所述的sgRNA向导序列,将各sgRNA向导序列的5’端添加CACC并去除3’端的PAM序列,若sgRNA 5’端第一个碱基不是G,则添加CACCG,形成正链sgRNA序列,将各sgRNA向导序列的反向互补序列的5’端添加碱基AAAC,形成负链sgRNA序列;
(2)载体构建:将所述的正链sgRNA序列和负链sgRNA序列退火形成如权利要求2中所述的三组双链DNA;将各组双链DNA分别与线性化的pGK1.1载体连接,得到连接产物并进行筛选获得三种阳性打靶载体即为靶向敲除猪GDPD2基因的敲除载体。
4.一种基于CRISPR/Cas9技术特异性靶向敲除猪GDPD2基因的细胞系,其特征在于,该细胞系为将权利要求3中所述的三种阳性打靶载体混合电转入IPEC-J2细胞并进行筛选和验证得到的。
5.一种基于CRISPR/Cas9技术靶向敲除猪小肠上皮细胞系中GDPD2基因的方法,其特征在于,该方法包括以下步骤:
(1)靶位点设计及sgRNA序列的合成:依据猪GDPD2基因CDS区所在的第一个外显子设计如权利要求1所述的sgRNA向导序列,将各sgRNA向导序列的5’端添加CACC并去除3’端的PAM序列,若sgRNA 5’端第一个碱基不是G,则添加CACCG,形成正链sgRNA序列,将各sgRNA向导序列的反向互补序列的5’端添加碱基AAAC,形成负链sgRNA序列;
(2)载体构建:将所述的正链sgRNA序列和负链sgRNA序列退火形成如权利要求2中所述的三组双链DNA;将各组双链DNA分别与线性化的pGK1.1载体连接,得到连接产物并进行筛选获得三种阳性打靶载体即为靶向敲除猪GDPD2基因的敲除载体;
(3)细胞转染:将三种阳性打靶载体混合电转染靶细胞IPEC-J2并进行筛选验证和培养获得猪GDPD2基因靶向敲除的IPEC-J2细胞。
6.根据权利要求5所述的方法,其特征在于,步骤(2)中得到连接产物并进行筛选获得三种阳性打靶载体的过程为:将连接产物转化大肠杆菌,挑取单克隆,摇菌培养后,根据sgRNA向导序列设计菌液PCR引物组,进行PCR筛选验证获得三种阳性打靶载体;所述菌液PCR引物组包含三对引物对,各引物对的正向引物为通用引物,反向引物分别为sgRNA反向互补序列,各引物对的具体序列如下所示:
F1:CATATGCTTACCGTAACTTGAAAG,
R1:CGGGACTCGGCCATGGTGAG;
F2:CATATGCTTACCGTAACTTGAAAG,
R2:CCCATACTTCACCTTGTTGG;
F3:CATATGCTTACCGTAACTTGAAAG,
R3:GTGGCAGCTATACAGACAGT。
7.根据权利要求5所述的方法,其特征在于,步骤(3)中所述筛选验证的过程为:
(Ⅰ)将三种阳性打靶载体混合电转染靶细胞IPEC-J2,经嘌呤霉素药筛,提取细胞基因组DNA,在敲除靶位点附近设计高特异性的引物进行PCR扩增,引物序列为:GDPD2-seqF:CCCCGGGCTCCTCAAATATC;GDPD2-seqR:TTCCTCCTGGGGAGAGAAAG;
(Ⅱ)CruiserTM酶切PCR扩增产物鉴定有效打靶的阳性克隆;
(Ⅲ)对CruiserTM酶切筛选的GDPD2基因敲除细胞进行TA克隆测序,获得GDPD2基因敲除的IPEC-J2细胞。
8.权利要求1中所述的sgRNA向导序列、权利要求2中所述的双链DNA、权利要求3中所述的敲除载体或权利要求4中所述的细胞系在猪抗病育种中的应用。
9.权利要求5所述的方法在猪抗病育种中的应用。
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