CN116376979A - 一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法 - Google Patents
一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法 Download PDFInfo
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Abstract
本发明涉及一种基于CRISPR‑Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,包括以下步骤:步骤1)、设计一种特异性靶向鸡CAR基因的sgRNA;步骤2)、构建一种用于靶向敲除鸡CAR基因的CRISPR‑Cas9系统,CRISPR‑Cas9系统中含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA,或者含有携带编码Cas9蛋白的编码序列和编码sgRNA的编码序列;CRISPR‑Cas9系统中,Cas9蛋白基因和sgRNA序列位于同一载体上,所述载体为lentiCRISPR v2;步骤3)、将lentiCRISPR v2质粒转染到细胞中,利用CRISPR‑Cas9系统编辑实现CAR基因沉默,通过药物筛选杀死阴性细胞,随后通过亚克隆的方法获得单克隆细胞株,从而获得鸡源CAR敲除的细胞系。本发明为探究CAR生物学功能与解析CAR基因在FAdV‑8b的感染中的作用打下了基础,具有较大的应用研究价值。
Description
技术领域
本发明涉及一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,属于基因工程技术领域。
背景技术
CRISPR-Cas系统是一种最早发现于古细菌的病毒防御机制系统,对病毒的感染和外来DNA的入侵起防御和保护作用。其中CRISPR-Cas9系统是目前应用最普遍的系统,它主要由RNA导向的Cas9核酸内切酶、一条导向RNA(sgRNA)和反式激活RNA(tracrRNA)组成。在该系统中,crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA结合形成双链RNA,并引导Cas9蛋白通过与目标序列3’端的PAM序列(通常为“NGG”)进行识别特异性地剪切双链DNA,造成DNA双链断裂(DSB,double strand break),细胞内启动的DNA损伤修复机制会导致修复后发生碱基缺失或插入,从而达到移码突变、沉默基因表达的效果,最终实现基因敲除的目的。目前该技术已经成熟地运用于真核细胞的基因组编辑中。
柯萨奇腺病毒受体(Coxsackie and adenovirus receptor,CAR)是柯萨奇B型病毒和腺病毒(Ad)共同受体,属于免疫球蛋白(Ig)超家族(IgSF)中的结合部黏附分子(JAM)家族,该蛋白定位于紧密连接处和上皮细胞侧膜。CAR的结构包括胞外区(V-type和C2-typeIg区)、跨膜区和胞质区。目前对CAR的功能认识不够完全,已明确的部分生物学功能包括:作为腺病毒受体、黏附蛋白、抑瘤作用、传递信号、免疫调节功能,且已有报道鸡源CAR是血清1型禽腺病毒(FAdV-1)和血清4型禽腺病毒(FAdV-4)的受体。本发明通过CRISPR-Cas9技术,药物筛选后通过亚克隆的方法,成功获得鸡源CAR敲除的细胞系(CAR-KO细胞系),且发现CAR-KO细胞系能够有效抑制FAdV-8b的感染。本发明为探究CAR生物学功能与解析CAR基因在FAdV-8b的感染中的作用打下了基础,具有较大的应用研究价值。
发明内容
本发明的目的是在于构建一个CAR-KO细胞系,提供一种基于CRISPR-Cas9编辑技术的CAR-KO细胞系的构建方法,具体为一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法。本发明的原理和最核心的关键技术是科学合理的构建了靶向鸡CAR基因的sgRNA,之后转染带有sgRNA和Cas9基因的lentiCRISPR v2质粒,利用CRISPR-Cas9系统达到基因沉默,通过药物筛选杀死阴性细胞,随后通过亚克隆的方法获得单克隆细胞株,从而获得CAR-KO细胞系。
本发明的目的是这样实现的:一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,包括以下步骤:
步骤1)、设计一种特异性靶向鸡CAR基因的sgRNA,所述的sgRNA位于鸡CAR基因的第五个外显子区域,且靶序列唯一;
步骤2)、构建一种用于靶向敲除鸡CAR基因的CRISPR-Cas9系统,CRISPR-Cas9系统中含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA,或者含有携带编码Cas9蛋白的编码序列和编码sgRNA的编码序列;CRISPR-Cas9系统中,Cas9蛋白基因和sgRNA序列位于同一载体上,所述载体为lentiCRISPR v2;
步骤3)、将步骤2)构建的含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA的lentiCRISPR v2质粒转染到细胞中,利用CRISPR-Cas9系统编辑实现CAR基因沉默,通过药物筛选杀死阴性细胞,随后通过亚克隆的方法获得单克隆细胞株,从而获得鸡源CAR敲除的细胞系。
步骤1)中,对鸡CAR基因设计了1条sgRNA,所述sgRNA的靶位点位于CAR基因的第五个外显子,该外显子序列如SEQ ID NO.1所示。
所述的特异性靶向鸡CAR基因的sgRNA编码链及互补链,其序列如SEQ ID NO.2所示。
步骤1)中,靶向鸡CAR基因的sgRNA制备方法,包括将合成的sgRNA编码链和互补链退火形成双链DNA,之后通过BsmBI限制性内切酶酶切载体,将双链DNA置于T7启动子之下构建获得。
步骤3)中,所述的细胞系为敲除鸡CAR基因的鸡肝癌细胞。
本发明先进科学,通过本发明提供的一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,本发明提供的一种用于靶向敲除鸡CAR基因的CRISPR-Cas9系统,在所述的系统中含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA,或者含有携带编码Cas9蛋白的编码序列和编码sgRNA的编码序列。其次,本发明所述的CRISPR-Cas9系统中,Cas9蛋白的编码序列与sgRNA的编码序列位于同一质粒上,所述的质粒为lentiCRISPR v2。
敲除CAR基因的具体操作如下:
(1)查找目的基因:利用NCBI在线数据库查找鸡CAR基因组序列NCBI ReferenceSequence:XM_416681.5,在该基因的第五个外显子设计敲除靶位点,第五个外显子的序列如SEQ NO.1所示。
(2)sgRNA的构建:设计好的sgRNA编码链和互补链分别命名为sgRNA-F和sgRNA-R,把引物稀释至100μM浓度,体系如下:sgRNA-F和sgRNA-R各1μL,PCR buffer 1μL,ddw 7μL,退火程序为5℃/min梯度降温至25℃,获得双链DNA。另一方面,利用BsmBI限制性内切酶酶切lentiCRISPR v2载体,酶切体系如下:BsmBI限制性内切酶1μL lentiCRISPR v2质粒1μg,0.1M DTT 0.5μL,NEB buffer3.1 5μL,其余用超纯水补至50μL,酶切条件为55℃30min,之后通过跑胶后胶回收获得含有粘性末端的lentiCRISPR v2载体质粒。最后,将获得的sgRNA与酶切后的载体质粒通过连接酶连接,最终将sgRNA放入lentiCRISPR v2质粒中,获得sgRNA和Cas9蛋白基因位于同一载体的质粒,命名为CAR-sgRNA。
(3)敲除细胞系的获得:6孔板中转染构建好的CAR-sgRNA,每孔4μg质粒,阴性对照转染pcDNA3.1质粒。48h后加入含有嘌呤霉素的培养基进行药物筛选,每隔2d换液,待阴性对照细胞全部死亡后,换成正常的培养基,存活的细胞生长至适当密度后,利用有限稀释法进行亚克隆至96孔板中,待细胞生长至足够数量时进行鉴定,之后冻存阳性细胞。
本发明的目的是构建一种基于CRISPR-Cas9靶向敲除鸡CAR基因的方法,基于本发明建立的CAR-KO细胞系可以有效抑制FAdV-8b的感染。本发明基于CRISPR-Cas9靶向CAR-KO细胞系构建在相关领域未见报道,本发明为探究CAR生物学功能与解析CAR基因在FAdV-8b的感染中的作用打下了基础,具有较大的应用研究价值。
附图说明
图1为lentiCRISPR v2载体质粒的酶切鉴定;
泳道M:Super DNA marker;泳道1:酶切后lentiCRISPR v2载体质粒。
图2为Western blot鉴定亚克隆的CAR-KO细胞系;
泳道M:Protein marker;泳道1:正常的鸡肝癌细胞系;泳道2:CAR-KO细胞系。
图3为亚克隆CAR-KO细胞系的测序鉴定。
图4为Western blot比较FAdV-8b在CAR-KO细胞系和野生型鸡肝癌细胞系中复制水平;
泳道1:野生型鸡肝癌细胞感染FAdV-8b;泳道2:CAR-KO细胞系感染FAdV-8b。
具体实施方式
1.查找目的基因:利用NCBI在线数据库查找鸡CAR基因组序列NCBI ReferenceSequence:XM_416681.5,在该基因的第五个外显子设计敲除靶位点,第五个外显子的序列如SEQ NO.1所示。
2.利用在线设计网站设计针对靶序列的sgRNA:利用张锋实验室网站https://zlab.bio/guide-design-resources进行sgRNA的设计,并在sgRNA-F的5’端前部添加CACCG,在sgRNA-R的5’端添加AAAC,3’端添加C,具体引物序列见表1,由苏州金唯智生物科技有限公司合成。
表1.靶向针对鸡CAR基因的sgRNA
3.双链sgRNA的获得:用ddw将sgRNA-F和sgRNA-R溶解,使其终浓度为100μM浓度,利用以下退火体系:sgRNA-F和sgRNA-R各1μL,10×PCR buffer 1μL,ddw 7μL,退火程序为5℃/min梯度降温至25℃,获得双链DNA。
4.载体质粒的酶切:利用BsmB I限制性内切酶酶切lentiCRISPR v2载体,酶切体系如下:BsmB I限制性内切酶1μL,lentiCRISPR v2质粒1μg,0.1M DTT 0.5μL,NEBbuffer3.1 5μL,其余用ddw补至50μL,酶切条件为55℃30min,之后通过跑凝胶电泳(图1),胶回收获得含有粘性末端的lentiCRISPR v2载体质粒。
5.表达sgRNA载体质粒的构建:将退火获得的双链sgRNA进行100倍稀释,同时将胶回收的载体质粒稀释至50ng/μL,之后按照以下体系进行连接:载体质粒1μL,双链sgRNA1μL,T4连接酶1μL,10×T4 buffer 1μL,ddw 6μL,在4℃连接过夜,随后转化至Stbl3感受态细胞中,挑取菌落提质粒后送测序鉴定。
6.敲除细胞系的筛选:准备一个6孔板的鸡肝癌细胞,每个孔分别转染6μg的sgRNA,阴性对照转染6μg的pcDNA3.1质粒,转染6h后换成10%生长液,48h后弃去原培养基,加入含有6μM嘌呤霉素的10%培养基进行筛选,每隔2d换液,待阴性对照组的细胞全部死亡后,加入无药物的10%生长液,当细胞密度达到80%左右时,进行亚克隆,同时收集细胞进行Western blot验证,选择保留sgRNA工作效率较高的亚克隆的细胞板。
7.敲除细胞系的鉴定:待亚克隆的细胞生长至足够密度时,传代至48孔板、24孔板、12孔板和6孔板,之后收集细胞进行Western blot鉴定和测序鉴定,以野生型鸡肝癌细胞为阴性对照,结果分别如图2和如图3;
8.将鸡肝癌细胞和CAR-KO细胞系感染MOI=0.1的FAdV-8b,细胞在感染48小时后(48hpi)进行Western blot分析,比较FAdV-8b在两种细胞系上复制的水平,结果如图4所示,感染FAdV-8b的正常鸡肝癌细胞(对照)中可以检测到明显的Hexon蛋白条带,与对照相比感染FAdV-8b的CAR-KO细胞系中检测到很浅的Hexon蛋白条带,表明所构建的CAR-KO细胞系有效抑制了FAdV-8b的感染复制。
SEQ ID NO.1
鸡CAR基因第五个外显子的核苷酸序列:
ACAAAAATACAGGGGAACTTCTCTTGAAAAATGCCTCTAAAGACTATTCTGGTACATACAGTTGTGTTGCTTCAAACCGAGTTGGCACAGATGAATGTTCTGTTGAGCTGAATGTCACCCCTC
Claims (5)
1.一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,包括以下步骤:
步骤1)、设计一种特异性靶向鸡CAR基因的sgRNA,所述的sgRNA位于鸡CAR基因的第五个外显子区域,且靶序列唯一;
步骤2)、构建一种用于靶向敲除鸡CAR基因的CRISPR-Cas9系统,CRISPR-Cas9系统中含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA,或者含有携带编码Cas9蛋白的编码序列和编码sgRNA的编码序列;CRISPR-Cas9系统中,Cas9蛋白基因和sgRNA序列位于同一载体上,所述载体为lentiCRISPR v2;
步骤3)、将步骤2)构建的含有Cas9蛋白和上述特异性靶向鸡CAR基因的sgRNA的lentiCRISPR v2质粒转染到细胞中,利用CRISPR-Cas9系统编辑实现CAR基因沉默,通过药物筛选杀死阴性细胞,随后通过亚克隆的方法获得单克隆细胞株,从而获得鸡源CAR敲除的细胞系。
2.根据权利要求1所述的一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,步骤1)中,对鸡CAR基因设计了1条sgRNA,所述sgRNA的靶位点位于CAR基因的第五个外显子,该外显子序列如SEQ ID NO.1所示。
3.根据权利要求2所述的一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,所述的特异性靶向鸡CAR基因的sgRNA编码链及互补链,其序列如SEQ ID NO.2、SEQ ID NO.3所示。
4.根据权利要求2所述的一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,步骤1)中,靶向鸡CAR基因的sgRNA制备方法,包括将合成的sgRNA编码链和互补链退火形成双链DNA,之后通过BsmBI限制性内切酶酶切载体,将双链DNA置于T7启动子之下构建获得。
5.根据权利要求2所述的一种基于CRISPR-Cas9编辑技术的敲除鸡柯萨奇腺病毒受体CAR基因的细胞系的构建方法,其特征在于,步骤3)中,所述的细胞系为敲除鸡CAR基因的鸡肝癌细胞。
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