CN113088521A - 一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法 - Google Patents
一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法 Download PDFInfo
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Abstract
本发明属于生物工程领域，涉及一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法，包括靶向小鼠Ahnak2基因的sgRNA载体构建、sgRNA的体外转录、F0代小鼠获取及鉴定、F1代杂合小鼠获取及鉴定、纯合后代的获取等过程构建Ahnak2基因敲除动物模型，所构建的动物模型，解决了传统基因敲除技术中基因脱靶率高、动物成活率低的问题，通过筛选更为高效的靶向小鼠Ahnak2基因的sgRNA载体，并对进一步降低脱靶率，提高小鼠后代阳性率及模型构建的成功率，经过6代纯合自交依然能够获得纯合后代，使得本发明方法构建的动物模型具有良好的稳定性，在心脏病、结直肠癌、子宫癌等肿瘤机理研究及药物开发领域具有重要应用价值。
Description
技术领域
本发明属于生物工程领域，具体涉及利用基因修饰技术制作基因敲出动物模型的领域，更具体涉及一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法。
背景技术
CRISPR/Cas系统是一种来源于细菌获得性免疫的由RNA介导Cas蛋白对目的基因进行靶向修饰的技术。经过研究者改造过的TypeⅡCRISPR/Cas9系统自2013年成功敲除哺乳动物细胞后，现在已经被应用于多种模式生物的基因敲除。CRISPR/Cas9系统载体构建简单快速，易操作，省时省力周期短，且几乎对所有物种都适用。CRISPR/Cas9和TALEN(Transcription Activator-like Effector Nucleases)的作用都是实现染色体上特定位点的双链断裂，然后引发自主损伤修复，修复会引发插入或缺失，从而造成基因序列永久的缺失，即基因敲除。针对每个基因，CRISPER/Cas9只需要构建一个sgRNA(singleguideRNA)，而且效率都很高，序列选择限制较小，只需要基因组上出现GG就可以。与zinc-finger nuclease(ZFN)和TALEN相比，CRISPER/Cas9引起的脱靶效应较高，但是使用成对sgRNA/Cas9-D10A>截短的sgRNA或者FoKI-dCas9可以极大的降低脱靶效应。目前，CRISPER/Cas9主要应用于基因定点突变(插入或缺失)、基因定点敲除、两位点同时突变、小片段的缺失、编码基因和非编码基因(lncRNA、microRNA)的靶向基因敲除。
Ahnakβ位于11q12.2，长1108bp，由6个外显子和5个内含子组成，其中开放阅读框(ORF)长450bp(301-750nt)，编码含有149个氨基酸，蛋白质大小约为16.0KD。它是Ahnak的第二个转录剪接体。第一个转录剪接体Ahnakα的cDNA为18815个碱基，编码一个680KD的巨型蛋白。通过BLAST比对，发明人所在研究团队首次发现Ahnakα与Ahnakβ的N端是一样的，但是Ahnakβ的C端与α完全不一样。因为已知Ahnakα具有控制心肌收缩的作用，提示Ahnakβ可能与心脏发育相关。研究表明Ahnak2基因在小鼠成体中心脏和肠的表达最强，其次在子宫的表达较强，在脾脏和肾有微弱的表达。因此，基于该前期研究成果构建Ahnak2基因敲除的动物模型，对于心脏病、结直肠癌、子宫癌的发病机理的研究及药物开发具有极为重要的意义。
发明内容
本发明的目的在于提供一种简单、高效且成功率高的基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法。
基于上述目的，本发明采用如下技术方案：
第一方面，本发明提供一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法，包括以下步骤：
S1：靶向小鼠Ahnak2基因的sgRNA载体构建
以Ahnak2内含子位置作为敲除区域，根据靶基因Ahnak2设计一对相应的sgRNA序列，sgRNA序列如SEQ ID NO.1和SEQ ID NO.2所示；
S2：sgRNA的体外转录
将步骤S1中合成的一对sgRNA和cas9 mRNA进行体外转录；
S3：F0代小鼠获取及鉴定
将步骤S2中获得的mRNA与cas9质粒一并通过显微注射的方式注入目标受精卵中，培育获得F0代小鼠；
将获得的F0代小鼠进行剪尾、genetyping鉴定，挑选Ahnak2基因敲出的阳性F0代小鼠；
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于性成熟后配繁，将出生的F1代小鼠进行剪尾、genetyping鉴定，挑选Ahnak2基因敲出的阳性F1代杂合小鼠；
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到Ahnak2基因敲除动物模型。
进一步地，sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对Ahnak2基因的sgRNA。
第二方面，本发明提供了一种由上述方法构建得到的Ahnak2基因敲除动物模型。
第三方面，本发明提供了一种利用CRISPR/Cas9技术进行Ahnak2基因敲除的试剂盒，该试剂盒包括sgRNA载体和用于检测Ahnak2基因的检测试剂；sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对Ahnak2基因的sgRNA。
进一步地，上述试剂盒还包括用于表达Cas9mRNA的Cas9表达质粒。
第四方面，本发明提供了上述Ahnak2基因敲除动物模型在制备治疗心脏病或癌症药物中的应用。
进一步地，在上述应用中，所述癌症包括结直肠癌和子宫癌。
与现有技术相比，本发明的有益效果如下：
本发明基于CRISPR/Cas9技术构建Ahnak2基因敲除的动物模型，解决了传统基因敲除技术中基因脱靶率高、动物成活率低的问题，本发明筛选更为高效的靶向小鼠Ahnak2基因的sgRNA载体，并对进一步降低脱靶率，提高小鼠后代阳性率及模型构建的成功率，经过6代纯合自交依然能够获得纯合后代，使得本发明方法构建的动物模型具有良好的稳定性，在心脏病、肿瘤机理研究及药物开发领域具有重要应用价值。
具体实施方式
实施例1
一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法，包括以下步骤：
S1：靶向小鼠Ahnak2基因的sgRNA载体构建
以Ahnak2内含子位置作为基因敲除区域，根据靶基因Ahnak2设计一对相应的sgRNA序列；BsaⅠ酶切pUC57-sgRNA载体，37℃水浴1h后，1％的琼脂糖电泳，回收酶切产物；然后将sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物；然后将sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物，转化大肠杆菌，挑选单克隆进行PCR，PCR结果呈阳性送测序验证，得到正确的sgRNA载体，其中sgRNA序列如下表1中的SEQ ID NO.1和SEQID NO.2所示。
表1基于Ahnak2内含子位置靶基因设计sgRNA的序列
| sgRNA名称 | SEQ ID NO | 序列(5’to3’) |
| Ahnak2-sgRNA1 | SEQ ID NO.1 | ACCACTCTTCCGCGGCCCTACGG |
| Ahnak2-sgRNA3 | SEQ ID NO.2 | GTGCACCAGGCGGGGTAGGTGGG |
S2：sgRNA的体外转录
将步骤S1中合成的一对sgRNA和cas9 mRNA，利用转录试剂盒进行体外转录；转录试剂盒为AM1354+AM1908，购自Ambion公司；含有cas9 mRNA的Cas9表达质粒为cas9 DA0A(plasmid#42335)，Addgene。
S3：F0代小鼠获取及鉴定
(1)准备单细胞受精卵
于性成熟小鼠腹腔注射马绒毛膜促性腺激素，注射量为5IU/只，46-48小时后注射人绒毛膜促性腺激素，注射完人绒毛膜促性腺激素后将2只雌鼠与单放雌鼠合笼。第四天上午检栓，见栓的记为0.5天；脱颈椎处死见栓0.5天的小鼠，剪出输卵管，用显微镊取出成团的卵子，透明质酸酶消化后，挑选形态饱满，胞质均匀的受精卵于M16中培养。
(2)显微注射受精卵
将挑选的受精卵转移入准备好的M2条带中，排成一列(每列30-50枚左右)，共注射180枚。将注射皿放在倒置显微镜的载物台上，使M2液滴长条的方向与操作者垂直，即位于y轴上。将注射管刺入胞浆内，注入步骤S2中获得的mRNA与cas9质粒即Cas9 sgRNA体系，见到胞质松散后迅速退针。注射结束后，将受精卵转移至含有M16培养液的培养皿中，放入37℃，5％二氧化碳培养箱恢复0.5-1.0小时。将受精卵移植到E0.5天假孕受体内。移植后大约19-21天出生F0代小鼠。
(3)F0代小鼠出生与鉴定
出生小鼠数量为9只，全部存活，将获得的F0代小鼠出生1周后进行剪尾、利用PCR进行genetyping鉴定，得到9只阳性F0代小鼠，毛色为黑色，性别为7雌2雄。
利用PCR进行genetyping鉴定的所用试剂及PCR程序如下表2、3和4所示。
表2 PCR反应体系
| 试剂 | 体积(微升) | 浓度 |
| 反应缓冲液(浓缩液，使用时作10倍稀释) | 2.5 | -- |
| 双蒸水 | 16.75 | -- |
| 上游引物 | 1 | 10微摩尔 |
| 下游引物 | 1 | 10微摩尔 |
| 镁离子溶液 | 2 | 25毫摩尔 |
| dNTPs | 0.5 | 10毫摩尔/样 |
| Taq DNA聚合酶 | 0.25 | 5酶活单位/微升 |
| 模板 | 1 | 100纳克/微升 |
表3 PCR进行genetyping鉴定过程中涉及到的引物
| 引物名称 | 引物序列 | 扩增片段长度 |
| Ahnak2-seq-F | CGTTCCTAGCCCTCAAATGTTCTCT | 野生型＝969bp |
| Ahnak2-seq-R | GAGGCGTTCCCACCTTAACAGTG | 基因敲除：450bp |
表4 PCR进行genetyping鉴定的程序
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于8周龄性成熟后配繁，将出生的F1代小鼠于1周龄进行剪尾、genetyping鉴定，具体鉴定方法参照S3中的方法，挑选Ahnak2基因敲出的阳性F1代杂合小鼠。
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到Ahnak2基因敲除动物模型。
由上述方法构建的Ahnak2基因敲除动物模型列表如表5所示，可以看出，由本发明所述方法构建的Ahnak2基因敲除动物模型，具有动物成活率高，且自交6代后依然能够获取纯合后代，具有良好的稳定性，便于进行心脏病和结直肠癌、子宫癌等肿瘤的机理研究以及相关药物开发。
表5小鼠模型构建过程中小鼠成活率及基因型
实施例2
本实施例提供了一种利用CRISPR/Cas9技术进行Ahnak2基因敲除的试剂盒，该试剂盒包括sgRNA载体、表达Cas9mRNA的Cas9表达质粒和用于检测Ahnak2基因的检测试剂；sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对Ahnak2基因的sgRNA。其中sgRNA的序列如SEQ ID NO.1和SEQ ID NO.2所示。
Claims (7)
1.一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法，包括以下步骤：
S1：靶向小鼠Ahnak2基因的sgRNA载体构建
以Ahnak2内含子位置作为敲除区域，根据靶基因Ahnak2设计一对相应的sgRNA序列，sgRNA序列如SEQ ID NO.1和SEQ ID NO.2所示；
S2：sgRNA的体外转录
将步骤S1中合成的一对sgRNA和cas9 mRNA进行体外转录；
S3：F0代小鼠获取及鉴定
将步骤S2中获得的mRNA与cas9质粒一并通过显微注射的方式注入目标受精卵中，培育获得F0代小鼠；
将获得的F0代小鼠进行剪尾、genetyping鉴定，挑选Ahnak2基因敲出的阳性F0代小鼠；
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于性成熟后配繁，将出生的F1代小鼠进行剪尾、genetyping鉴定，挑选Ahnak2基因敲出的阳性F1代杂合小鼠；
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到Ahnak2基因敲除动物模型。
2.根据权利要求1所述的构建方法，其特征在于，所述sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对Ahnak2基因的sgRNA。
3.一种由权利要求1或2所述方法构建得到的Ahnak2基因敲除动物模型。
4.一种利用CRISPR/Cas9技术进行Ahnak2基因敲除的试剂盒，其特征在于，所述试剂盒包括sgRNA载体和用于检测Ahnak2基因的检测试剂；所述sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对Ahnak2基因的sgRNA。
5.根据权利要求3所述的试剂盒，其特征在于，所述试剂盒还包括用于表达Cas9mRNA的Cas9表达质粒。
6.权利要求3所述Ahnak2基因敲除动物模型在制备治疗心脏病或癌症药物中的应用。
7.根据权利要求6所述的应用，其特征在于，所述癌症包括结直肠癌和子宫癌。
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| CN114958907A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9技术构建Bcl6基因超级增强子敲除动物模型的方法及应用 |
| CN114958908A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 |
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| CN114958907A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9技术构建Bcl6基因超级增强子敲除动物模型的方法及应用 |
| CN114958908A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 |
| CN115011637A (zh) * | 2021-12-31 | 2022-09-06 | 河南省人民医院 | 基于CRISPR/Cas9技术构建LCR基因敲除动物模型的方法及其应用 |
| CN114958908B (zh) * | 2021-12-31 | 2024-03-29 | 河南省人民医院 | 基于CRISPR/Cas9构建Ets2基因超级增强子敲除动物模型的方法及其应用 |
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