CN113621610B - 一对敲除猪HSPA6基因部分保守区域的sgRNA序列及应用 - Google Patents
一对敲除猪HSPA6基因部分保守区域的sgRNA序列及应用 Download PDFInfo
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Abstract
本发明提供一对敲除猪HSPA6基因部分保守区域的sgRNA序列及应用,应用在敲除HSPA6基因部分保守基因区域中,或应用在制备敲除HSPA6基因部分保守区域产品中的应用,本发明能实现有效敲除,并且敲除后显著抑制猪HSPA6基因表达,本发明应用于以HSPA6基因为靶点的神经退行性疾病及应激性疾病药物的研发,构建的敲除HSPA6基因部分保守区域的猪源细胞可作为解析HSPA6基因表达的分子调节机制的研究模型。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一对敲除HSPA6基因部分保守区域的sgRNA序列及应用。
技术背景
随着位点特异性核酸酶的发展,CRISPR/Cas9因其简单、高效、低成本、多功能性和特异性而开始对生物学研究产生革命性的影响。CRISPR/cas9介导的基因组工程已被广泛高效地应用于各种物种中。这种功能强大的基因编辑工具可实现条件性基因敲除、基因敲入、基因替换、点突变等等,为基因的功能研究提供了一个有吸引力的策略。
HSPA6基因是一种仅在高等哺乳动物中发现的新进化的HSP70家族的基因。HSPA6基因具有严格诱导性,其基础表达水平较低或不表达,而在特定应激源的刺激下,其表达水平会迅速且显著地上升。基于该基因独特的基因表达模式,已构建了一种能有效将应激信号转变为EGFP荧光信号的报告细胞系,即细胞传感器。HSPA6基因也参与人类神经元细胞对蛋白毒性应激的反应,对神经退行性疾病有影响。除此之外,HSPA6基因在动物应激性疾病中也有重要的作用。而目前关于HSPA6基因分子调控机制方面的研究很有限,因此,本研究基于已有的工作基础和技术平台,以CRISPR/Cas9介导的基因敲除技术探究敲除保守区对HSPA6基因表达的影响。
发明内容
本发明阐述了一段能促进猪HSPA6基因表达保守区域,该部分保守区域被敲除后能显著抑制猪HSPA6基因表达,能敲除该部分保守区域的物质可用于启动子区调控元件解析、细胞传感器和神经退行性疾病及应激性疾病药物靶点研究,所述的物质应用于敲除HSPA6基因部分保守区域或应用在制备敲除HSPA6基因部分保守区域的产品中,所述的部分保守区域的DNA序列的正义链如SEQ ID NO:5所示,其对应互补链的DNA序列如SEQ ID NO:6所示。
本发明提供的所述的物质为一对敲除猪HSPA6基因部分保守区域的sgRNA序列,所述的敲除基于CRISPR/Cas9***进行,其特征在于:所述的sgRNA序列包括sgRNA 2-1和sgRNA 2-2;
所述的sgRNA 2-1的DNA序列的正义链如SEQ ID NO:1所示,其对应互补链的DNA序列如SEQ ID NO:2所示;
所述的sgRNA 2-2的DNA序列的正义链如SEQ ID NO:3所示,其对应互补链的DNA序列如SEQ ID NO:4所示。
本发明还提供一种试剂盒,所述的试剂盒包含所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列。
本发明还提供两种可打靶猪HSPA6基因部分保守区域的CRISPR/Cas9***敲除载体,由所述的sgRNA 2-1和sgRNA 2-2的双链DNA与线性化的PX330载体连接得到,其制备方法是:首先设计合成靶向所述猪HSPA6基因部分保守区域的正链sgRNA序列和负链sgRNA序列;再将所述的正链sgRNA序列和负链sgRNA序列退火形成所述的双链DNA;最后将所述的双链DNA与载体连接得到打靶载体。
本发明还提供所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在制备HSPA6基因表达被显著抑制猪肾细胞中的应用,猪肾细胞在敲除所述的HSPA6基因部分保守区域后发生缺失,在加热后HSPA6基因表达量为239.02,相较于对照组细胞HSPA6基因的表达量4569.12,下降了94.77%,经过统计学计算,其p值为0.001,小于极显著阈值0.01,由此可知,HSPA6基因表达被极显著抑制。
本发明还提供所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在制备HSPA6基因表达被显著抑制猪模型中的应用。
本发明还提供所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在以HSPA6基因为靶点的神经退行性疾病及应激性疾病药物中的应用。
有益效果如下:
本发明发现了一段缺失后能显著抑制猪HSPA6基因表达的启动子区的部分保守区域,并且提供一对能有效敲除该部分保守区域的sgRNA序列,并验证了使用该sgRNA序列敲除部分保守区域实现了显著抑制猪HSPA6基因的表达的效果,本发明应用于以HSPA6基因为靶点的神经退行性疾病及应激性疾病药物的研发,构建的敲除HSPA6基因部分保守区域的猪源细胞可作为解析HSPA6基因表达的分子调节机制的研究模型。
附图说明:
图1为人、牛、羊、马、猪、川金丝猴和***骆驼的HSPA6基因启动子序列比对图;
图2为2对不同sgRNA切割效率的测序峰图;
图3为阳性猪肾细胞克隆鉴定的电泳图;
图4为克隆鉴定测序峰图
图5为EGFP定点整合的细胞克隆的PCR鉴定电泳图;
图6为本发明所述的部分保守区对猪HSPA6基因表达的调控功能的研究;
图7为加热处理后阳性克隆HSPA6及EGFP的mRNA表达量分析图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、根据所述的HSPA6基因部分保守区域设计sgRNA序列及构建打靶载体。
根据人、牛、羊、马、猪、川金丝猴和***骆驼的HSPA6基因第一外显子前3000bp的序列,七种哺乳动物的HSPA6基因启动子序列比对如图1所示,通过Vector软件进行多序列比对,得到一段71bp的能极显著影响猪HSPA6基因表达的部分启动子保守区域,所述的部分保守区域的DNA序列的正义链如SEQ ID NO:5所示,其对应互补链的DNA序列如SEQ IDNO:6所示。
根据所述的部分保守区域设计并合成了2对猪HSPA6部分启动子保守区域的sgRNA序列,其中一对为sgRNA 2-1和sgRNA 2-2;所述的sgRNA 2-1的DNA序列的正义链如SEQ IDNO:1所示,其对应互补链的DNA序列如SEQ ID NO:2所示;所述的sgRNA2-2的DNA序列的正义链如SEQ ID NO:3所示,其对应互补链的DNA序列如SEQ ID NO:4所示。另一对为sgRNA 1-1和sgRNA 1-2,所述的sgRNA 2-1的DNA序列的正义链如SEQ ID NO:7所示,其对应互补链的DNA序列如SEQ ID NO:8所示;所述的sgRNA 1-2的DNA序列的正义链如SEQ ID NO:9所示,其对应互补链的DNA序列如SEQ ID NO:10所示。
再将8条单链的sgRNA的DNA序列分别经过退火后形成4条靶向保守区不同位点的sgRNA的寡核苷酸链;然后将该寡聚核苷酸连入PX330质粒载体。所述的4条靶向保守区不同位点的sgRNA的寡核苷酸链为:
SgRNA-1-1序列:5-CACCGCATTGGCCGGGAATCGAACC-3;
SgRNA-1-1作用位点的序列:5-AAACGGTTCGATTCCCGGCCAATGC-3;
SgRNA-1-2序列:5-CACCGTGAACCACCAATGCATCTTG-3;
SgRNA-1-2作用位点的序列:5-AAACCAAGATGCATTGGTGGTTCAC-3;
SgRNA-2-1序列:5-CACCGATTGTCACAAATCCTAGTAG-3;
SgRNA-2-1作用位点的序列:5-AAACCTACTAGGATTTGTGACAATC-3;
SgRNA-2-2序列:5-CACCGTTAACATCATCCCTAGCCCT-3;
SgRNA-2-2作用位点的序列:5-AAACAGGGCTAGGGATGATGTTAAC-3。
实施例2、构建敲除HSPA6基因部分保守区域的猪胚胎成纤维细胞,评估和筛选sgRNA。
通过对4条sgRNA的表达载体进一步的测序后,进行质粒的大提和质粒的乙醇沉淀,将纯化后一定浓度的四种sgRNA的PX330的表达载体,通过电穿孔转染的方式引入到猪的胚胎成纤维细胞中,转染后的84小时,提取各组细胞的基因组,然后用特异性的检测突变效率的引物进行PCR反应,将所获得的PCR产物一方面送去测序,用于评估2对不同sgRNA切割效率的测序峰图如图2所示,图2中红色部分为PAM序列,黑色部分为sgRNA 1-1和sgRNA1-2,蓝色部分为sgRNA 2-1和sgRNA 2-2,虚框中为被敲除序列,其中绿色部分为启动子上的一段保守区域。
通过分析初步评估每对sgRNA的切割效率,从图2的测序峰图中可以看出sgRNA 1-1和sgRNA 1-2这对序列并不能有效敲除基因保守区域,而sgRNA 2-1和sgRNA 2-2有效敲除了基因保守区域,因此选择sgRNA 2-1和sgRNA 2-2作为后续实验的一对sgRNA序列。
实施例3、PX330质粒的转染。
复苏基于猪HSPA6的传感器细胞系,传代2-3次,然后用PBS洗2~3遍后消化该细胞,离心沉淀细胞后弃上清,加入电转染缓冲液,然后将两种PX330质粒按比例加入到细胞和缓冲液中,用移液器轻轻混匀后,轻轻的将混合液移入电极杯中,将电极杯放到电穿孔仪器上进行电击操作。电击完成后,将电极杯在室温静置10分钟后,将电极杯中的混合液转入细胞培养皿中。最后将该细胞培养皿置于37℃二氧化碳培养箱中培养。培养12小时后,换液。
实施例4、阳性克隆的筛选与鉴定
电转染48h后,通过极限稀释的方法将基于猪HSPA6的传感器细胞系铺到100mm细胞培养皿中,2~3天更换一次细胞培养液。9~10天后待细胞克隆长成后,将细胞放在42℃的细胞培养箱中进行热刺激处理1h,然后放在37℃的培养箱中适应1h后,接着在荧光显微镜下将与WT细胞发光强弱不同的细胞克隆统一做上标记,然后将这些标记过的克隆挑取入24孔细胞培养板中接着培养。2~3天后,待24孔板中的细胞长至一定得汇合度,对细胞进行传代同时分出部分克隆的细胞,将这些细胞用NP40裂解液裂解后再通过PCR和测序的方法进一步克隆鉴定及验证定点EGFP的整合事件。
从阳性细胞克隆鉴定的电泳图3可以看出,WT细胞的序列为624bp,而经过敲除后得到阳性克隆的序列为438bp。其中克隆细胞A1为纯合子,A2、A3、B1和C1为杂合子。
对这五种克隆细胞进行测序后得到图4。如图所示,蓝色部分为sgRNA序列,红色部分为PAM序列。红色箭头所指为切割位置。从图中可以看出,相比WT的序列,五种克隆细胞的序列都对保守区域进行了敲除。
图5为EGFP定点整合的细胞克隆的PCR鉴定电泳图,从图5中可以看出WT细胞和五种克隆细胞均有EGFP基因的敲入,可用于后续荧光分析。
实施例4保守区对猪HSPA6基因表达的调节功能的研究
对细胞克隆A1、A3、B1进行42℃加热刺激处理,刺激实验的结果如图6所示,在对保守区敲除之后,荧光强度有不同程度的变弱。通过q-PCR对结果进一步定量分析,发现在mRNA水平上,一些阳性克隆的HSPA6及EGFP的表达水平有了极显著性的下降。
图6包括WT和三种克隆的荧光分析图和实时定量图,根据图3的基因型将5种克隆分为3类,以A1、A3和B1为例。从图6的荧光图中可以看出3种克隆细胞的荧光强度明显弱于WT细胞。从实时定量图可以看出,42℃加热后WT和3种克隆细胞的HSPA6基因表达量分别为4569.12、239.02、2560.81、1345.26。相比于WT的表达量,3种克隆的表达量分别降低了94.44%、43.95%、70.56%。经过统计学分析,其p值分别为0.001、0.024、0.004。因此,A3可显著性降低HSPA6基因的表达量,而A1和B1极显著性降低HSPA6基因的表达量。而EGFP基因有和HSPA6基因相似的表达模式。
对加热处理后阳性克隆HSPA6及EGFP的mRNA表达量分析可,从图7以看出,在37℃时,WT和3种克隆细胞的表达量无显著性差异,与A2有显著性差异(p=0.02);与C1有极显著性差异(p=0.0001)。虽然表达量有差异,但相差倍数在5倍以内,因此可看作在37℃时,细胞之间无表达差异且HSPA6基因基本无表达。而在42℃加热后,WT和5种细胞的HSPA6基因mRNA表达量均有上升。相比于WT细胞HSPA6基因的表达量,A1、A2、A3、B1和C1的下降比例分别为94.44%、52.55%、43.95%、70.56%、99.28%。EGFP和HSAP6基因mRNA表达量有相同的表达模式。
在敲除了HSPA6基因启动子上的一段保守区域以后,该基因的mRNA表达量有极显著性降低,可推断该保守区域对HSPA6基因的表达有重要调控作用。
SEQUENCE LISTING
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Claims (6)
1.一对敲除猪HSPA6基因部分保守区域的sgRNA序列,所述的敲除基于CRISPR/Cas9***进行,其特征在于:所述的sgRNA序列包括sgRNA 2-1和sgRNA 2-2;
所述的sgRNA 2-1的DNA序列的正义链如SEQ ID NO:1所示,其对应互补链的DNA序列如SEQ ID NO:2所示;
所述的sgRNA 2-2的DNA序列的正义链如SEQ ID NO:3所示,其对应互补链的DNA序列如SEQ ID NO:4所示。
2.一种试剂盒,其特征在于:所述的试剂盒中包含如权利要求1所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列。
3.一种可打靶猪HSPA6基因部分保守区域的CRISPR/Cas9***敲除载体,其特征在于:由如权利要求1所述的sgRNA 2-1和sgRNA 2-2的双链DNA与载体连接得到打靶载体。
4.如权利要求1所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在制备HSPA6基因表达被显著抑制猪肾细胞中的应用。
5.如权利要求1所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在制备HSPA6基因表达被显著抑制猪模型中的应用。
6.如权利要求1所述的一对敲除猪HSPA6基因部分保守区域的sgRNA序列在制备以HSPA6基因为靶点的神经退行性疾病及应激性疾病药物中的应用。
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