CN105462968A - 一种靶向apoCⅢ的CRISPR-Cas9系统及其应用 - Google Patents
一种靶向apoCⅢ的CRISPR-Cas9系统及其应用 Download PDFInfo
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Abstract
本发明提供一种用于预防和/或治疗高血脂等心血管疾病的CRISPR-Cas9系统及其制备方法和用途。所述CRISPR-Cas9系统包括特异性靶向人apoCIII基因上特定区域的sgRNA序列和包含sgRNA序列的中间载体，所述apoCIII基因上的特定区域选自apoCIII基因外显子。本发明还提供了所述CRISPR-Cas9系统在制备用于预防和/或治疗高血脂等心血管疾病的药物用途。利用本发明制备的特异性靶向人apoCIII基因的sgRNA能够精确靶向人apoCIII基因并且实现基因敲除。该制备方法步骤简单、sgRNA靶向性好，CRISPR-Cas9系统的敲除效率高。
Description
技术领域
本发明涉及基因工程领域，更具体地说涉及CRISPR-Cas9特异性敲除人apoCIII基因的方法以及用于特异性靶向apoCIII基因的sgRNA。
背景技术
，高脂血症(Hyperlipidemia)是以血浆中胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)升高或高密度脂蛋白胆固醇(HDL-C)降低为表现的一种脂质代谢紊乱状态，是导致动脉粥样硬化的危险因素之一，可累及全身的很多重要脏器，引起冠心病、脑栓塞、下肢间歇性跛行等疾病。
载脂蛋白CIII(ApolipoproteinCIII，apoCIII)是一种水溶性低分子量(8.8kDa)蛋白质，主要由肝脏合成，是一种分泌型肝蛋白。载脂蛋白CIII是高密度脂蛋白(HDL)和富甘油三酯(TG)脂蛋白的成分。apoCIII的主要生理功能是抑制脂蛋白酯酶(lipoproteinlipase，LPL)活性和干扰脂蛋白与细胞表面葡糖胺聚糖基质结合。已知脂蛋白酯酶(LPL)是催化富含三酰甘油脂蛋白分解的关键酶。apoCIII能够阻碍脂蛋白上的apoE与低密度脂蛋白(LDL)及其相关蛋白受体的相互作用进而减少肝脏对富含三酰甘油脂蛋白的摄取。此外，apoCIII还能够抑制肝酯酶(HL)的活性。因此，apoCIII的升高导致TG水平升高，进而引起诸如高甘油三酯血症、心血管疾病等。已有研究发现，apoCIII基因中外显子3编码的N末端肽段是apoCIII抑制LPL活性的主要部位。目前，已有他汀类药物、苯氯酸等来调节肝脏分泌apoCIII，高三酰甘油血症和心血管病患者需长期服用，容易产生耐药性。靶向apoCIII基因位点的CRISPR-Cas9系统可以特异性地降低apoCIII的表达水平，促进富含三酰甘油脂蛋白的分解，进而达到降低血脂、预防和/或治疗高血脂等心血管疾病的目的。
CRISPR-Cas9是一种具有核酸内切酶活性的复合体，能够识别特定的DNA序列，进行特定位点切割从而造成双链DNA断裂，在没有模板的条件下，发生非同源重组末端连接，造成移码突变，导致基因敲除。该系统的主要组成部分为：sgRNA序列，负责靶向特异性基因位点；Cas9酶，负责对靶向位点的DNA进行修饰切割。这一技术具有更快速、简便、高效、多位点、特异性靶向敲除基因的优势。将该技术应用于靶向apoCIII基因的敲除，为实现高脂血症和心血管疾病的治疗提供了一种可能的选择。本发明的目的就是要验证利用CRISPR-Cas9高效靶向敲除apoCIII基因相应的技术方案，以达到特异性敲除apoCIII基因的目的。
发明内容
本发明的目的在于提供靶向apoCIII基因的CRISPR-Cas9系统及其制备方法和用途。
为达到以上目的，本发明采用以下技术方案：
一种利用CRISPR-Cas9系统在HepG2细胞中实现apoCIII基因敲除的方法，包括以下步骤：
1、根据apoCIII基因的序列设计和选择用于特异性靶向目的基因的sgRNA；
2、在目的基因apoCIII的sgRNA的基础上设计sgRNA双链寡聚核苷酸序列；
3、将目的基因apoCIII的sgRNA双链寡聚核苷酸序列与线性化的质粒载体连接，转化提取得到目的基因的sgRNA表达载体；
4、构建Cas9真核表达载体；
5、转染细胞获得apoCIII基因敲除细胞；
6、蛋白质印迹法(Westernblotting)检测apoCIII蛋白的表达。
上述技术方案所述的利用CRISPR-Cas9系统在HepG2细胞中实现apoCIII基因敲除的方法，其中，步骤1中，sgRNA在目的基因上的靶序列符合5′-GGN(19)GG或者5′-GN(20)GG或者5′-N(21)GG的序列排序规则。
再进一步地，所述步骤1中，sgRNA位于目的基因的3个外显子(如序列表SEQIDNO.1、2和3所示)上。
再进一步地，所述步骤1中，sgRNA位于目的基因的第2个(如序列表SEQIDNO.2所示)和第3个外显子(如序列表SEQIDNO.3所示)。
再进一步地，所述步骤1中，每条sgRNA的长度为16-22bp。
上述技术方案所述的利用CRISPR-Cas9系统在HepG2细胞中实现apoCIII基因敲除的方法，其中，步骤2中，sgRNA双链寡聚核苷酸序列的构建过程为：
(1)在sgRNA的5′端加上TAAG得到正向寡核苷酸(Forwardoligo)；
(2)再根据选择的sgRNA，获得其对应的DNA互补链，并在其5′端加上TAGG得到反向寡核苷酸(Reverseoligo)；
(3)分别合成上述的正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸的forwardoligo和reverseoligo成对变性、退火，退火之后形成可以连入pL-CRISPR.EFS.GFP载体的双链sgRNA寡聚核苷酸，如下：
本发明还提供了特异性靶向apoCIII基因的sgRNA，其序列如序列表SEQIDNO.4-40任意一条序列所示。该sgRNA可以单独应用于apoCIII基因的敲除，也可以多条sgRNA序列结合起来应用于apoCIII基因的敲除。
本发明还提供了一种能够在HepG2细胞中实现apoCIII基因敲除的CRISPR-Cas9系统在制备用于预防和/或治疗高血脂等心血管疾病的药物的用途。
与现有技术相比，本发明至少具有以下优点：目前治疗高血脂等心血管疾病的小分子药物或抗体药物均需要心血管病患者长期服用，易产生耐药性。本发明的CRISPR-Cas9系统优化设计针对apoCIII基因的3个外显子的sgRNA，能够高效抑制apoCIII基因的表达，从而降低血脂水平，有助于心血管疾病康复并有效降低高血脂症患者的心血管疾病的发病率。
附图说明
图1pL-CRISPR.EFS.GFP质粒结构
图2hCAS9质粒结构
图3Westernblotting检测apoCIII蛋白的表达
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。
实施例1
CRISPR-Cas9特异性敲除人apoCIII基因中用于特异性靶向apoCIII基因的sgRNA的设计和合成
1.靶向人apoCIII基因的sgRNA的设计
(1)在apoCIII基因上选择5′-GGN(19)GG或者5′-GN(20)GG或者5′-N(21)GG的序列。
(2)sgRNA在apoCIII基因上的靶向位点位于基因的外显子。
(3)sgRNA在apoCIII基因上的靶向位点位于不同的各种剪切形式的共有外显子上。
(4)在UCSC数据库中用BLAT或NCBI数据库中用BLAST，确定sgRNA的靶序列是否唯一，减少潜在的脱靶位点。
根据以上方法，我们一共设计了37个靶向人apoCIII基因的sgRNA，序列分别如序列表SEQIDNO.4-40所示。
2.靶向人apoCIII基因的sgRNA的选择
(1)靶向apoCIII基因的sgRNA的靶序列在apoCIII基因上不能离ATG起始子太近，防止转录后从下游另一个ATG开始而出现一个被截短的基因形式，不能保证基因完全失活。
(2)sgRNA在apoCIII基因上的靶向位点位于整个基因的中段。
(3)在apoCIII基因上选择相隔一定距离成对的位点。这样有利于形成特异性的片段缺失，也有利于降低脱靶效应。
根据以上方法，在37个靶向人apoCIII基因的sgRNA(序列分别如序列表SEQIDNO.4-40所示)中符合要求的序列有8个(分别如序列表SEQIDNO.10、12、13、14、16、18、19和20所示)，由于序列较多，没有必要一一进行实验验证，我们从中随机选取了2个(分别如序列表SEQIDNO.12和13所示)进行后续实验。
3.靶向人apoCIII基因的sgRNA寡聚核苷酸的合成和构建
根据选择的2个sgRNA(分别如序列表SEQIDNO.12和13所示)，在其5′端加上TAAG得到正向寡核苷酸(Forwardoligo)；根据选择的sgRNA，获得其对应DNA的互补链，并且在其5′端加上TAGG得到反向寡核苷酸(Reverseoligo)。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸的forwardoligo和reverseoligo成对变性、退火，退火后形成可以连入pL-CRISPR.EFS.GFP载体的双链sgRNA寡聚核苷酸。
变性、退火体系(10μl)为：
退火温度为55-60℃。
选择的第1个sgRNA(如序列表SEQIDNO.12所示)，其forwardoligo和reverseoligo成对变性、退火，退火后形成可以连入载体的双链sgRNA寡聚核苷酸。
选择的第2个sgRNA(如序列表SEQIDNO.13所示)，其forwardoligo和reverseoligo成对变性、退火，退火后形成可以连入载体的双链sgRNA寡聚核苷酸。
实施例2
利用CRISPR-Cas9特异性敲除人apoCIII基因(用于靶向apoCIII基因的sgRNA序列如序列表SEQIDNO.12所示)
1.线性化质粒pL-CRISPR.EFS.GFP的酶切，酶切完成后用AxyPrepPCRCleanupKit(AP-PCR-250)纯化回收至20-40μl灭菌水中。
2.将变性、退火之后获得的可以连入载体的双链sgRNA寡聚核苷酸与线性化pL-CRISPR.EFS.GFP相连获得pL-CRISPR.EFS.GFP-sgRNA质粒。
3.将上述步骤获得的连接产物转化到感受态细胞中并涂Amp+平板(50μg/μl)，并挑取克隆。
4.用通用引物测序来鉴定阳性克隆。
5.37℃摇床摇菌过夜培养阳性克隆，提取质粒，获得pL-CRISPR.EFS.GFP-sgRNA质粒。
6.细胞培养与转染。
7.新霉素筛选单克隆细胞株。
实施例3
利用CRISPR-Cas9特异性敲除人apoCIII基因(用于靶向apoCIII基因的sgRNA序列如序列表SEQIDNO.13所示)
1.线性化质粒pL-CRISPR.EFS.GFP的酶切，酶切完成后用AxyPrepPCRCleanupKit(AP-PCR-250)纯化回收至20-40μl灭菌水中。
2.将变性、退火之后获得的可以连入载体的双链sgRNA寡聚核苷酸与线性化pL-CRISPR.EFS.GFP相连获得pL-CRISPR.EFS.GFP-sgRNA质粒。
3.将上述步骤获得的连接产物转化到感受态细胞中并涂Amp+平板(50μg/μl)，并挑取克隆。
4用通用引物测序来鉴定阳性克隆。
5.37℃摇床摇菌过夜培养阳性克隆，并用试剂盒抽提质粒，获得pL-CRISPR.EFS.GFP-sgRNA质粒。
6.细胞培养与转染。
7.新霉素筛选单克隆细胞株。
实施例4
蛋白质印迹法(Westernblotting)检测apoCIII蛋白
1.对蛋白进行定量，然后取等量蛋白进行12％的SDS-PAGE电泳。
2.电泳结束后，将蛋白电转印至PVDF膜上，用封闭液封闭过夜。
3.次日用PBS在室温下脱色摇床洗涤3次，每次10min，加入ab21032抗体，4℃孵育过夜。
4.次日在室温下脱色摇床洗涤3次，每次10min，加入辣根过氧化物酶标记羊抗兔二抗，室温孵育1h；
5.PBS室温下脱色摇床洗涤3次，每次10min，最后用DAB对其进行染色。
经Westernblotting检测实施例2、3中的单克隆细胞株，均未发现apoCIII蛋白，说明本系统能够有效的敲除apoCIII基因。
Claims (10)
1.在CRISPR-Cas9特异性敲除人apoCIII基因中用于特异性靶向apoCIII基因的sgRNA，其特征在于，所述sgRNA在apoCIII基因上的靶序列位于基因的外显子且位于不同剪切形式的共有外显子上，靶序列唯一。
2.根据权利要求1所述的sgRNA，其特征在于，其对应的DNA序列如序列表SEQIDNO.4-40任意一条序列所示。
3.根据权利要求1所述的sgRNA，其特征在于，其对应的DNA序列如序列表SEQIDNO.10、12、13、14、16、18、19或者20任意一条序列所示。
4.根据权利要求1所述的sgRNA，其特征在于，其对应的DNA序列如序列表SEQIDNO.12或者13任意一条序列所示。
5.根据权利要求1-4任一项所述的CRISPR-Cas9系统，其特征在于，所述CRISPR-Cas9系统还包括Cas9。
6.根据权利要求5所述的CRISPR-Cas9系统，其特征在于，所述Cas9和所述特异性靶向apoCIII基因上的特定基因位点的sgRNA可以单独应用于apoCIII基因的敲除，也可以多个sgRNA应用于apoCIII基因的敲除。
7.根据权利要求5所述的CRISPR-Cas9系统，其特征在于，所述Cas9和所述特异性靶向apoCIII基因上的特定基因位点的sgRNA分别存在于质粒中或存在于同一质粒。
8.根据权利要求1-7任一项所述的制备方法，其特征在于，还包括将各sgRNA片段分别构建入质粒载体的步骤。
9.根据权利要求1-7任一项所述的CRISPR-Cas9系统在制备apoCIII基因敲除的细胞模型和动物模型中的用途。
10.根据权利要求1-7任一项所述的CRISPR-Cas9系统在制备用于预防和/或治疗高血脂等心血管疾病的药物的用途。
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