CN113774060A - 基于CRISPR-Cas系统构建GSTZ1基因敲除细胞系的方法及其应用 - Google Patents
基于CRISPR-Cas系统构建GSTZ1基因敲除细胞系的方法及其应用 Download PDFInfo
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Abstract
本发明涉及基因工程领域,具体公开了一种基于CRISPR‑Cas系统构建GSTZ1基因敲除细胞系的方法及其应用。本发明基于CRISPR‑Cas系统,成功以人正常肝细胞GSTZ1基因为靶标,获得了高效、特异、精准靶向人正常肝细胞GSTZ1基因的sgRNA分子,如SEQ ID NO:1~SEQ ID NO:2所示,能够靶向人正常肝细胞GSTZ1基因,从而引导Cas蛋白对GSTZ1基因的特异性切割,最终达到敲除GSTZ1基因的目的;并且本发明基于CRISPR/Cas‑sgRNA表达系统敲除GSTZ1基因能够影响肝细胞的增殖。
Description
技术领域
本发明涉及基因工程领域,尤其是涉及一种基于CRISPR-Cas系统构建GSTZ1基因敲除细胞系的方法及其应用。
背景技术
肝脏作为重要的组成器官,在新陈代谢、蛋白质合成和排毒中起着核心作用,受伤时具有独特的再生能力。尽管已经确定了许多在肝脏修复过程中调节细胞增殖的因素,但受伤的肝脏在组织恢复之前维持重要功能的机制尚不清楚。近年来由于肝病极高的发病率,严重影响到了病人的生活,因此研究者们力争利用动物模型去寻求治疗肝损伤的办法。探索肝损伤的机理,使其对于临床上肝病的防治,发病机制的研究和护肝药物的筛选有着重要意义。
谷胱甘肽S-转移酶ζ1(GSTZ1)是近年来发现的谷胱甘肽硫转移酶(GSTs)超家族新成员之一,也被称为马来酰乙酰乙酸异构酶(MAAI),主要存在于肝脏和肾脏内、一些植物、真菌和哺乳动物内。人谷胱甘肽S-转移酶ζ1(h-GSTZ1)基因长度大约为10.9kb,有9个外显子和8个内含子构成,定位在人类染色体14q24.3,由216个氨基酸组成,分子量在24kDa左右。GSTZ1主要有2大生物学功能,其一可以参与苯丙氨酸和酪氨酸的分解代谢,催化马来酰乙酰乙酸(MAA),转化为延胡索酰乙酰乙酸(FAA);其二催化二氯乙酸盐(DCA)、氟乙酸盐、二氯丙酸酯与GSH加成。
CRISPR/Cas9系统是从细菌和古生菌对抗外来病毒或质粒的适应性免疫系统发展而来,包括三种不同的类型,其中TypeⅡ型的CRISPR/Cas系统的DNA内切酶Cas9只有一个亚基,结构最为简单,所以应用也最广泛。除了Cas9蛋白外,该系统还包括两条短的CRISPRRNAs(crRNAs)和trans-activating crRNAs(tracrRNA)。由于CRISPR/Cas9系统具有操作简单,切割效率高等特点,被认为有更好的应用前景。
目前还没有人公开敲除人正常肝细胞GSTZ1基因是否会影响肝细胞增殖等相关研究。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种基于CRISPR-Cas系统构建GSTZ1基因敲除细胞系的方法及其应用。本发明中sgRNA可以高效靶向人正常肝细胞GSTZ1基因,制备的GSTZ1基因敲除细胞系会影响肝细胞的增殖,促进肝细胞的活力。
为实现上述目的,本发明采取的技术方案为:
第一目的,本发明提供了一种靶向人正常肝细胞GSTZ1基因的sgRNA,所述sgRNA由sgRNA-F和sgRNA-R组成,sgRNA-F的核苷酸序列如SEQ ID NO:1所示,sgRNA-R的核苷酸序列如SEQ ID NO:2所示。
采用Ensembl和Crispr设计工具及sgRNA的设计原则设计得到上述的sgRNA。
第二目的,本发明提供了一种CRISPR/Cas慢病毒载体,包含上述的靶向人正常肝细胞GSTZ1基因的sgRNA。
作为本发明所述CRISPR/Cas慢病毒载体的优选实施方式,所述CRISPR/Cas慢病毒载体由慢病毒载体lentilCRISPR v2经Esp3I酶切后,连入带Esp3I粘性末端的上述的靶向人正常肝细胞GSTZ1基因的sgRNA重组得到。
第三目的,本发明提供了一种慢病毒,含有上述的CRISPR/Cas慢病毒载体。
第四目的,本发明提供了一种基于CRISPR-Cas系统构建人正常肝细胞GSTZ1基因敲除细胞系的方法,包括以下步骤:
1)在sgRNA-F的5’端加上CACC得到正向寡核苷酸,在sgRNA-R的5’端加上AAAC得到反向寡核苷酸,分别合成正向寡核苷酸和反向寡核苷酸,将正向寡核苷酸和反向寡核苷酸变性、退火,形成双链DNA片段;
2)将步骤1)得到的双链DNA片段与Cas载体连接,得到重组表达载体;
3)将步骤2)得到的重组表达载体与包装系统共转染包装细胞,收获病毒后纯化、浓缩,得到病毒颗粒;
4)将步骤3)制得的病毒颗粒感染细胞,筛选稳定转染的细胞,得到成功敲除GSTZ1基因的细胞系。
作为本发明所述方法的优选实施方式,步骤2)中Cas载体为慢病毒载体lentilCRISPR v2。
作为本发明所述方法的优选实施方式,步骤3)中包装细胞为293T细胞;
作为本发明所述方法的优选实施方式,步骤3)中病毒颗粒包括上述的sgRNA。
更优选地,包装系统中的包装载体为psPAX2和/或Pmd.2G。
第五目的,本发明提供了上述方法制备的敲除GSTZ1基因的细胞系。
第六目的,本发明提供了上述细胞系在肝细胞增殖检测中的应用。
更优选地,肝细胞增殖检测包括CCK8、克隆形成等实验。
通过对本发明敲除GSTZ1基因的细胞系进行功能分析,发现敲除GSTZ1基因后可以影响肝细胞的增殖,为肝损伤研究奠定基础。
与现有技术相比,本发明具有以下有益效果:
1)本发明基于CRISPR-Cas系统,成功以人正常肝细胞GSTZ1基因为靶标,获得了高效、特异、精准靶向人正常肝细胞GSTZ1基因的sgRNA分子,能够靶向人正常肝细胞GSTZ1基因,从而引导Cas蛋白对GSTZ1基因的特异性切割,最终达到敲除GSTZ1基因的目的;
2)通过本发明基于CRISPR/Cas-sgRNA表达系统敲除GSTZ1基因能够影响肝细胞的增殖。
附图说明
图1为实施例3中利用293T细胞进行慢病毒包装24h后的100X显微观察图;
图2为实施例3中利用Puromycin抗性筛选LO2细胞48h后的显微图;
图3为本发明敲除GSTZ1基因的细胞系的Western Blot结果图;
图4为本发明敲除GSTZ1基因的细胞系的CCK8细胞增殖检测曲线图;
图5为本发明敲除GSTZ1基因的细胞系的克隆形成结果图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、CRISPR/Cas9基因敲除载体构建
1、靶向人正常肝细胞GSTZ1基因的sgRNA的选择和设计
在Genebank中找到人GSTZ1的基因序列,通过Crispr设计工具及sgRNA的设计原则,评估GSTZ1序列上得分较高的靶位点设计sgRNA,其评估原则包括:1)序列下游是否有PAM(NGG),2)脱靶效率低,不易脱靶。
sgRNA-F:GCCCAGAACGCCATCACTTG(SEQ ID NO:1);
sgRNA-R:CGGGTCTTGCGGTAGTGAAC(SEQ ID NO:2);
在sgRNA-F的5’端加上CACC得到正向寡核苷酸,在sgRNA-R的5’端加上AAAC得到反向寡核苷酸,分别合成正向寡核苷酸和反向寡核苷酸,将正向寡核苷酸和反向寡核苷酸变性、退火,形成双链DNA片段。
2、将双链DNA片段和用Esp3I酶切后的慢病毒载体lentilCRISPR v2进行连接,体系为:4μL DNA,1μL lentilCRISPR v2,1μL T4 DNA连接酶,2μL T4DNA连接酶buffer,12μL无酶水。16℃连接过夜,将连接产物转化到大肠杆菌DH5a感受态细胞中,涂布于带有氨苄青霉素抗性的LB平板上,筛选阳性菌落,提取阳性菌落质粒进行分析及测序,确定CRISPR/Cas9基因敲除载体构建成功(命名为V2-GsgRNA)。
实施例2、细胞培养
293T细胞和LO2细胞复苏的具体步骤为;
1.当细胞传代次数过多,细胞状态变差时,或者细胞出现污染事故时,需要丢弃并对最初冻存的细胞进行复苏;
2.设置温度为37℃的水浴;
3.查看细胞库记录,根据记录从液氮罐中取出冻存的细胞(需戴上棉手套,防止被冻伤),迅速丢入水浴锅中并快速晃动,尽量在1~2min内使细胞溶液完全溶解;
4.将细胞溶液转移到15ml离心管中,并在其中加上2ml新鲜的完全培养基,混匀后离心,1000rpm,3min;
5.去掉上清,加入5ml新鲜的完全培养基,混匀沉淀后,转入6cm培养皿;
6.将培养皿平稳放入37℃、5%CO2和95%相对湿度的培养箱中培养;
7.第二天观察细胞存活率,给细胞换一下培养基,以后每天观察细胞生长情况,为后边实验提供状态良好的细胞。
嘌呤霉素筛选LO2细胞的具体步骤为:
1.首先将上述已复苏状态良好的LO2细胞铺在6孔板中:
2.至细胞长到70%,分别用0.5ug/ml、1ug/ml、1.5ug/ml、2ug/ml、2.5ug/ml、3ug/ml、3.5ug/ml、4ug/ml、4.5ug/ml、5ug/ml的嘌呤霉素加到野生型的LO2细胞中,12h、24h分别看细胞的死亡状态;
3.24h后发现4.5ug/ml孔中的细胞大部分死亡,48h后该孔中的细胞彻底死亡;
4.确定了4.5ug/ml浓度的嘌呤霉素是LO2细胞筛选的最佳浓度。
实施例3、慢病毒包装及其感染
具体步骤为:
1、在转染前将293T细胞铺在中皿中,等到密度70%-80%时进行转染。
2、以V2-GsgRNA:psPAX2:pMD2.G=4:3:1的比例进行慢病毒包装,48h后将获得的慢病毒上清液,3000转离心5分钟,用0.45μm的滤膜过滤上清后得病毒颗粒,分装冻存于-80℃备用。其中慢病毒包装24h后100X显微观察图如图1所示。
3、在感染前一天将LO2细胞铺在6孔板中,等到密度为70%-80%时进行感染;
4、移除细胞培养基,加入步骤2备好的病毒颗粒;
5、感染6-8小时后移除慢病毒,换成新鲜无抗生素培养基培养24小时后,加入4.5ug/ml puromycin杀死未被感染的细胞,每隔24小时换一次液;其中,采用Puromycin抗性筛选LO2细胞48h后的显微图如图2所示。
将挑取的单克隆细胞利用15%的胎牛血清培养基扩增培养,提取细胞蛋白并用Western Blot检测GSTZ1表达情况,提取GSTZ1表达缺失的单克隆细胞系基因组DNA,进行pcr,进行TA克隆并送检测。鉴定到一株单克隆细胞系,命名为GSTZ1 KO。
实施例4、Western Blot对单克隆细胞敲除实验
具体步骤为:
1.制备浓度为10%的SDS凝胶;
2.将凝胶安装到电泳夹上,并固定到电泳槽中,先向两胶板间倒入电泳液,检测是否漏液,再加满电泳液;
3.轻柔的拔去梳子,去除孔中气泡,按顺序加样;
4.恒压80V电泳30min左右,待溴酚蓝移动至分离胶与积层胶交界处时,转换电压为120V,电泳1h,至溴酚蓝离胶底1cm左右停止电泳;
5.根据预染蛋白Marker,切下目的条带,浸入转膜液中;根据胶块大小,裁剪PVDF膜,甲醇中活化3min,浸入转膜液中;于转膜液中按照滤纸-胶-PVDF膜-滤纸的三明治结构将胶块和PVDF膜装配进转膜夹中,使胶位于黑色阴极,PVDF膜位于白色阳极,并将转膜夹安装到转膜仪中,黑色面对黑色,白色面对红色;
6.将转膜仪固定到电泳槽中,加入-20℃预冷的转膜液,将整个装置于冰浴中转膜;转膜条件为恒流200mA 2.5h;
7.转膜结束,用镊子夹取PVDF膜,于TBST中漂洗几下,用5%BSA室温封闭2h;
8.用一抗稀释液稀释一抗至适当浓度。封闭结束后,将PVDF膜在TBST中漂洗几下,于4℃孵育一抗过夜;
9.TBST洗膜5min×4次;
10.二抗室温孵育1h;
11.TBST洗膜5min×4次;
12.避光条件下配制ECL工作液,等体积混合A、B液;用滤纸吸干膜上的TBST,然后将ECL工作液滴加到PVDF膜上,室温避光孵育3min,在凝胶成像系统中曝光(本发明敲除GSTZ1基因的细胞系的Western Blot结果图如图3所示)。
用Western Blot检测克隆细胞系GSTZ1蛋白的表达,发现GSTZ1蛋白表达缺失。
实施例5、CCK8细胞增殖检测实验
将敲除GSTZ1基因的细胞系进行CCK8细胞增殖检测实验。
具体步骤为:
1.采用0.25%胰酶-EDTA消化细胞,制成单细胞悬液,血球计数板计数细胞浓度。采用全培调整细胞浓度至1×104个/ml;
2.将单细胞悬液以100ul/孔加入96孔细胞培养板中,每个样品六个重复;
3.同时以全培为空白对照,分别在0h,6h,12h,24h和48h分别每孔加入10ulCCK8溶液,37℃避光孵育0.5h;
4.用酶标仪测定450nm处的吸光度,每个重复测定三次;
5.用细胞的吸光度扣除空白孔的吸光度,得到相对吸光度值;
6.采用SPSS 24.0做统计分析并用Graphpad prism 5软件绘制生长曲线(本发明敲除GSTZ1基因的细胞系的CCK8细胞增殖检测曲线图如图4所示)。结果显示敲除GSTZ1后能够促进肝细胞的增殖能力。
实施例6、克隆形成实验
具体步骤为:
1.采用0.25%胰酶-EDTA消化细胞,制备单细胞悬液;
2.采用血球计数板计数后以1000个/孔将细胞均匀铺到六孔板中,每三天换一次液;
3.待细胞长至肉眼可见克隆时,去除培养基;
4.采用PBS轻柔清洗细胞两次后用4%多聚甲醛固定细胞15min;
5.弃去多聚甲醛,用清水清洗细胞表面;
6.晾干后用亚甲蓝染色10min,用清水洗净染液;
7.于体视显微镜下计数细胞数大于50的单克隆数量(本发明敲除GSTZ1基因的细胞系的克隆形成结果如图5所示)。克隆形成实验表明,GSTZ1基因敲除可以明显促进肝细胞的增殖。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 五邑大学
<120> 基于CRISPR-Cas系统构建GSTZ1基因敲除细胞系的方法及其应用
<130> 2021.08.24
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> 人工合成
<400> 1
gcccagaacg ccatcacttg 20
<210> 2
<211> 20
<212> DNA
<213> 人工合成
<400> 2
cgggtcttgc ggtagtgaac 20
Claims (10)
1.一种靶向人正常肝细胞GSTZ1基因的sgRNA,其特征在于,所述sgRNA由sgRNA-F和sgRNA-R组成,sgRNA-F的核苷酸序列如SEQ ID NO:1所示,sgRNA-R的核苷酸序列如SEQ IDNO:2所示。
2.一种CRISPR/Cas慢病毒载体,其特征在于,包含如权利要求1所述的靶向人正常肝细胞GSTZ1基因的sgRNA。
3.如权利要求2所述的CRISPR/Cas慢病毒载体,其特征在于,所述CRISPR/Cas慢病毒载体由慢病毒载体lentilCRISPR v2经Esp3I酶切后,连入带Esp3I粘性末端的权利要求1所述的靶向人正常肝细胞GSTZ1基因的sgRNA重组得到。
4.一种慢病毒,其特征在于,含有权利要求2或3所述的CRISPR/Cas慢病毒载体。
5.一种基于CRISPR-Cas系统构建人正常肝细胞GSTZ1基因敲除细胞系的方法,其特征在于,包括以下步骤:
1)在权利要求1所述的sgRNA-F的5’端加上CACC得到正向寡核苷酸,在权利要求1所述的sgRNA-R的5’端加上AAAC得到反向寡核苷酸,分别合成正向寡核苷酸和反向寡核苷酸,将正向寡核苷酸和反向寡核苷酸变性、退火,形成双链DNA片段;
2)将步骤1)得到的双链DNA片段与Cas载体连接,得到重组表达载体;
3)将步骤2)得到的重组表达载体与包装系统共转染包装细胞,收获病毒后纯化、浓缩,得到病毒颗粒;
4)将步骤3)制得的病毒颗粒感染细胞,筛选稳定转染的细胞,得到成功敲除GSTZ1基因的细胞系。
6.如权利要求5所述的方法,其特征在于,步骤2)中Cas载体为慢病毒载体lentilCRISPR v2。
7.如权利要求5所述的方法,其特征在于,步骤3)中包装细胞为293T细胞。
8.如权利要求5所述的方法,其特征在于,步骤3)中病毒颗粒包括如权利要求1所述的sgRNA。
9.如权利要求5~8任一项所述的方法制备的敲除GSTZ1基因的细胞系。
10.如权利要求9所述的细胞系在肝细胞增殖检测中的应用。
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CN1852974A (zh) * | 2003-06-09 | 2006-10-25 | 密歇根大学董事会 | 用于治疗和诊断癌症的组合物和方法 |
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Title |
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JINGJING LI等: "GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway", 《JOURNAL OF EXPERIMENTAL AND CLINICAL CANCER RESEARCH》 * |
雷冲等: "基于CRISPR-Cas9系统构建GSTZ1基因敲除模型并探索其对肝癌细胞增殖和迁移能力的影响", 《重庆医科大学学报》 * |
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