CN107287245B - 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 - Google Patents
一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。包括以下步骤:一、靶向小鼠Glrx1基因的gRNA的选择和设计。二、sgRNA载体构建。三、sgRNA体外转录。四、小鼠一细胞期受精卵注射。五、F0代小鼠出生和鉴定。六、阳性F0代小鼠配繁,F1代小鼠出生与鉴定。通过本发明方法,能够成功获得Glrx1基因敲除动物模型。
Description
技术领域
本发明属于利用基因修饰技术制作基因敲除动物模型的领域,具体涉及一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。
背景技术
CRISPR/Cas(Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated)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Type II CRISPR/Cas9系统自2013年成功敲除哺乳动物细胞后,现在己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,CRISPR/Cas9只需要构建一个sgRNA(single guideRNA),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-fingernucleases(ZFNs)and TALEN相比,CRISPR/Cas9系统具有相同或者更高的基因编辑效率,更便宜。相对于TALEN,CRISPR/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,CRISPR/Cas9主要应用于基因定点突变(插入或缺失)、基因定点敲入、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。
谷氧还蛋白(glutaredoxin,Glrx)普遍存在于细菌、病毒和哺乳动物体内,其表达受干扰素(interferon,IFN)调控,分子量为12kDa,由106-107个氨基酸残基组成,是硫氧还蛋白(thioredoxin,Trx)家族的重要分支,作为电子供体,参与组成巯基-二硫键氧化还原酶家族,依靠谷胱甘肽(GSH)将氧化状态的蛋白质二硫键还原为巯基,以维持细胞氧化还原稳态,在细胞信号转导过程中发挥重要作用。有文献报道,在氧化应激引起的损伤中,蛋白质氧化损伤先于核酸,蛋白发生羰基化和糖基化,从而失去生物活性。大量研究表明Glrx1是一种具有多种生物学功能的多效性细胞因子,与调节氧化还原反应、细胞生长和抑制凋亡有密切关系,与人类某些疾病,如获得性免疫缺陷综合征和细菌感染等的发生、发展也相关。
谷氧还蛋白是机体内能特异、高效的还原谷胱甘肽化蛋白质的一种酶蛋白,Glrx特异的恢复氧化应激损伤产生的谷胱甘肽化蛋白活性的能力可能会使其成为热点药物。构建Glrx1基因敲除小鼠模型,对于研究氧化应激、营养健康等具有重要意义。但传统的基因敲除方法成功率极低,一直未得到应用。近年来,CRISPR/Cas9技术得到广泛应用,为Glrx1基因敲除模型鼠的构建及其在营养与健康研究中的应用提供了可能性。
发明内容
本发明的目的是提供一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法。
本发明的目的通过以下技术方案实现。
一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法,包含以下步骤:
步骤一:靶向小鼠Glrx1基因的gRNA的选择和设计
在Glrx1内含子相应位置设计相应的sgRNA,其引物序列如SEQ ID NO.1和SEQ IDNO.2所示;
步骤二:sgRNA载体构建
首先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,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;
步骤三:采用转录试剂盒,体外转录sgRNA和Cas9 mRNA,转录好的sgRNA备用;试剂盒名称:AM1354+AM1908,Ambion by Life Technologies;
步骤四:Cas9 sgRNA体系(Cas9 mRNA和sgRNA)的受精卵显微注射;Cas9表达质粒为cas9 D10A(plasmid#42335),Addgene;
步骤五:F0代小鼠出生与鉴定;
步骤六:F0小鼠性成熟配繁,F1代小鼠鉴定。
其中个,步骤六优选:F0代小鼠在性成熟后和C57BL/6J小鼠回交进行配繁,出生F1代小鼠在1周龄进行剪尾鉴定,得到阳性的F1代杂合子。
进一步优选从mRNA水平和酶解测序来鉴定F1代。
一种基于CRISPR-Cas9基因敲除技术的Glrx1基因敲除试剂盒,包括:
1)sgRNA载体,所述的sgRNA载体以pUC57-sgRNA载体为出发载体,含针对Glrx1基因的sgRNA;该sgRNA由SEQ ID NO.1和SEQ ID NO.2所示的sgRNA引物退火得到;
2)以及配套的检测试剂,用于检测Glrx1基因的剪切效果和评估基因敲除效率。
本发明所述的基于CRISPR-Cas9基因敲除技术的Glrx1基因敲除试剂盒,优选还包含Cas9 mRNA或用于表达Cas9 mRNA的Cas9表达质粒。
有益效果:
本实验难点其一在于sgRNA序列定位,本实验采用的sgRNA序列高效,不易脱靶;其二在于对Cas9 sgRNA体系的优化,使小鼠后代阳性率更高,脱靶率低。应用该技术制作的Glrx1敲除小鼠解决了传统基因敲除技术中基因脱靶率高、动物成活率低等瓶颈问题,可广泛应用在膳食营养与健康、氧化应激及相关疾病的研究中的应用。
附图说明
图1、Glrx-1-Cas9-KO小鼠策略设计图
图2、sgRNA载体图谱
图3、PCR检测策略
图4、61#,62#,64#电泳结果
图5、73#,74#,75#电泳结果
图6、7周龄雄性纯合Glrx1-/-小鼠照片
具体实施方式
实施例1
基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法通过以下步骤实现:
步骤一:靶向小鼠Glrx1基因的gRNA的选择和设计
设计Glrx-1-Cas9-KO小鼠策略,如图1所示。根据策略,设计相应sgRNA序列,根据策略,在Glrx-1内含子相应位置设计相应的sgRNA,订购相应Oligo;sgRNA序列如下:
sgRNA名称 | 序列 | PAM |
Glrx-3S1(forward) | CGGAGATGACACTTACTGATGGG(SEQ ID NO.1) | GGG |
Glrx-5S1(forward) | GCTAAGCGCCGCTGCATTACCGG(SEQ ID NO.2) | CGG |
步骤二:sgRNA载体构建
首先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。然后将订购的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,载体图谱如图2所示。
步骤三:sgRNA体外转录
采用转录试剂盒,体外转录sgRNA和Cas9 mRNA,转录好的sgRNA和Cas9 mRNA备用。试剂盒名称:AM1354+AM1908,购自Ambion公司.
步骤四:受精卵显微注射
1.准备单细胞受精卵
小鼠超排:第一天,腹腔注射马绒毛膜促性腺激素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只雌鼠与单放雄鼠合笼。第四天上午检栓,见栓的记为0.5天。
获取受精卵: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、胞质均匀的胚胎于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(30-50枚左右)。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Cas9 sgRNA体系(sgRNA和Cas9 mRNA),Cas9表达质粒为cas9 D10A(plasmid#42335),Addgene;见到胞质松散后迅速退针。注射结束后,将胚胎转移至含有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。
步骤五:F0代小鼠出生与鉴定
出生小崽数量为39只,存活数量38只,F0代小鼠出生1周后进行剪尾鉴定,得到7只阳性F0代小鼠,毛色为黑色,性别为5雌2雄,图6为F1代两个雄性纯合Glrx1-/-小鼠照片。
PCR反应体系:
PCR检测策略如图3所示。
步骤六:F0小鼠性成熟配繁,F1代小鼠鉴定
F0代小鼠在8周龄左右性成熟和C57BL/6J小鼠回交进行配繁,出生F1代小鼠在1周龄进行剪尾鉴定,得到6只阳性的F1代杂合子,列表如下:
序号 | 性别 | 颜色 | 基因型 | 雌/雄 | 代数 |
61 | ♂ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
62 | ♂ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
64 | ♀ | 黑 | -7588bp/wt,E1-E2(整个编码区)全部删除 | ♂14 | F1 |
73 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
74 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
75 | ♀ | 黑 | -7898bp/wt,E1-E2(整个编码区)全部删除 | ♀7 | F1 |
分别从mRNA水平和酶解测序来鉴定F1代,mRNA水平所采用qPCR手段来实现,条件同上。从测序结果可看出61#,62#,64#,73#,74#,75#与野生型对比序列长度至少少了-7588bp,也就是说明E1-E2被删掉。
61#,62#,64#:
GCCCTTTAAAACTGAAGCATCCTACTTGGTAACTCCTCCTCCAAGGAGGTTCCTTATTAAATGAGAGCTGCTGGCTAAGCGCC--------------7588bp----------ATACACATAGTTCTAGACATAAATACACAAAAAGATAACGT
73#,74#,75#:
CCAGTGTGCAATGGTAGGCCTAGGAAGTACTGACTCATACCAA--------------7898bp---------TAGCTAAGGATGGAAATTTGGGAAGTAT
实施例2
本实施例与实施例1的不同点在于步骤三中所用单链DNA模板和引物序列为2074-Glrx-gtF1。其它步骤与实施例1相同;结果均与实施例1相同。
实施例3
本实施例与实施例1的不同点在于步骤四中合笼雄鼠的品种优选为C57BL/6J雄鼠。其它步骤与实施例1相同。
<110> 南京农业大学
<120> 一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法
<160> 2
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物Glrx-3S1
<400> 1
cggagatgac acttactgat ggg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<220>
<223> 引物Glrx-5S1
<400> 2
gctaagcgcc gctgcattac cgg 23
Claims (5)
1.一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法,其特征在于包含以下步骤:
步骤一:靶向小鼠Glrx1基因的sgRNA的选择和设计
在Glrx1内含子相应位置设计相应的sgRNA,所述sgRNA引物的核苷酸序列如SEQ IDNO.1和SEQ ID NO.2所示;
步骤二:sgRNA载体构建
首先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;然后将步骤一中所述的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;
步骤三:采用转录试剂盒,体外转录sgRNA和Cas9 mRNA,转录好的sgRNA备用;其中,Cas9表达质粒为Addgene plasmid # 42335;
步骤四:Cas9 mRNA和sgRNA 组成的Cas9 sgRNA体系的小鼠受精卵显微注射,其中,Cas9表达质粒为Addgene plasmid # 42335;
步骤五:F0代小鼠出生与鉴定;
步骤六:F0小鼠性成熟配繁,F1代小鼠鉴定。
2.根据权利要求1所述的构建方法,其特征在于步骤六:F0代小鼠在性成熟后和C57BL/6J小鼠回交进行配繁,出生F1代小鼠在1周龄进行剪尾鉴定,得到阳性的F1代杂合子。
3.根据权利要求2所述的构建方法,其特征在于从mRNA水平和酶解测序来鉴定F1代小鼠。
4.一种基于 CRISPR-Cas9 基因敲除技术的Glrx1基因敲除试剂盒,其特征在于包括 :
1) sgRNA载体,所述的sgRNA载体以pUC57-sgRNA载体为出发载体,含针对Glrx1基因的sgRNA;该sgRNA由SEQ ID NO.1和SEQ ID NO.2所示的sgRNA引物退火得到;
2)以及配套的检测试剂,用于检测 Glrx1基因的剪切效果和评估基因敲除效率。
5.根据权利要求4所述的基于 CRISPR-Cas9 基因敲除技术的Glrx1基因敲除试剂盒,其特征在于还包含Cas9 mRNA或用于表达Cas9 mRNA的Cas9表达质粒。
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Attenuated cardiovascular hypertrophy and oxidant generation in response to angiotensin II infusion in glutaredoxin-1 knockout mice;Markus M.Bachschmid等;《Free Radical Biology & Medicine》;20101015;第49卷(第7期);第1221-1229页,参见摘要 * |
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GB2578026A (en) | 2020-04-15 |
CN107287245A (zh) | 2017-10-24 |
GB201918320D0 (en) | 2020-01-29 |
WO2018219093A1 (zh) | 2018-12-06 |
GB2578026A8 (en) | 2020-07-22 |
GB2578026B (en) | 2022-11-23 |
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