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WO2018219093A1 - 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 PDF

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WO2018219093A1
WO2018219093A1 PCT/CN2018/085432 CN2018085432W WO2018219093A1 WO 2018219093 A1 WO2018219093 A1 WO 2018219093A1 CN 2018085432 W CN2018085432 W CN 2018085432W WO 2018219093 A1 WO2018219093 A1 WO 2018219093A1
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sgrna
cas9
glrx1
mice
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李春保
周光宏
邹小雨
石学彬
徐幸莲
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Nanjing Agricultural University
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Definitions

  • the invention belongs to the field of making a gene knockout animal model by using a genetic modification technology, and particularly relates to a method for constructing a Glrx1 gene knockout animal model based on CRISPR/Cas9 technology.
  • the CRISPR/Cas (Clustered Regularly Interspaced Shot Palindromic repeats/CRISPR-associated) system is a technique for RNA-mediated Cas protein targeted modification of a gene of interest derived from bacterial acquired immunity.
  • the Type II CRISPR/Cas9 system which has been modified by researchers, has been successfully knocked out of mammalian cells since 2013 and has now been used for gene knockout of multiple model organisms.
  • the CRISPR/Cas9 system vector is simple, fast, easy to operate, time-saving and labor-saving, and is suitable for almost all species.
  • CRISPR/Cas9 and TALEN Transcription Activator-like Effector Nucleases
  • TALEN Transcription Activator-like Effector Nucleases
  • CRISPR/Cas9 only needs to construct a single sgRNA (single guide RNA), and the efficiency is very high, the sequence selection restriction is small, only GG is needed in the genome.
  • ZFNs zinc-finger nucleases
  • TALEN Transcription Activator-like Effector Nucleases
  • CRISPR/Cas9 Compared to TALEN, CRISPR/Cas9 caused a higher off-target effect, but the use of paired sgRNA/Cas9-D10A> truncated sgRNA or FoKI-dCas9 can greatly reduce the off-target effect.
  • CRISPR/Cas9 is mainly used for targeted site knockout of gene site-directed mutagenesis (insertion or deletion), gene-spotted knock-in, simultaneous two-point mutation, deletion of small fragments, coding genes and non-coding genes (lncRNA, microRNA). .
  • Glutaredoxin (Glrx) is ubiquitous in bacteria, viruses and mammals. Its expression is regulated by interferon (IFN), its molecular weight is 12kDa, it is composed of 106-107 amino acid residues, and it is thioredox.
  • IFN interferon
  • Trx thioredoxin
  • Glrx1 is a pleiotropic cytokine with a variety of biological functions, which is closely related to the regulation of redox reaction, cell growth and inhibition of apoptosis, and certain diseases of humans, such as acquired immunodeficiency syndrome and The occurrence and development of bacterial infections are also relevant.
  • Glutathione is an enzyme protein that specifically and efficiently reduces glutathionylated proteins in the body. Glrx's ability to restore glutathionylated protein activity caused by oxidative stress damage may make it a hot spot. drug. Construction of the Glrx1 knockout mouse model is of great significance for the study of oxidative stress, nutritional health and so on. However, the traditional gene knockout method has a very low success rate and has not been applied. In recent years, CRISPR/Cas9 technology has been widely used, providing possibilities for the construction of Glrx1 knockout model mice and their application in nutrition and health research.
  • the object of the present invention is to provide a method for constructing a Glrx1 knockout animal model based on CRISPR/Cas9 technology.
  • a method for constructing a Glrx1 knockout animal model based on CRISPR/Cas9 technology comprising the following steps:
  • Step 1 Selection and design of gRNA targeting mouse Glrx1 gene
  • Step 2 sgRNA vector construction
  • BsaI was digested with pUC57-sgRNA vector. After 1 h of water bath at 37 °C, 1% agarose was electrophoresed to recover the digested product; then the sgRNA primer was annealed; finally, the annealed product and the recovered digested product were ligated and transformed into E. coli. Select the monoclonal for PCR, and the PCR result is positive and sent to the sequencing to verify that the correct sgRNA vector is obtained;
  • Step 3 In vitro transcription of sgRNA and Cas9 mRNA using a transcription kit, and transcription of a good sgRNA for use; kit name: AM1354+AM1908, Ambion by Life Technologies;
  • Step 4 Microinjection of fertilized eggs of Cas9sgRNA system (Cas9 mRNA and sgRNA); Cas9 expression plasmid is cas9D10A (plasmid #42335), Addgene;
  • Step 5 Birth and identification of F0 generation mice
  • Step 6 F0 mice were sexually matured and matured, and F1 mice were identified.
  • step 6 F0 generation mice are backcrossed with C57BL/6J mice after sexual maturation, and F1 generation mice are tail-tailed at 1 week of age to obtain positive F1 heterozygotes.
  • a Glrx1 gene knockout kit based on CRISPR-Cas9 gene knockout technology comprising:
  • an sgRNA vector comprising a pUC57-sgRNA vector as a starting vector, comprising an sgRNA targeting a Glrx1 gene; the sgRNA is annealed by an sgRNA primer represented by SEQ ID NO. 1 and SEQ ID NO. 2;
  • the CRISPR-Cas9 gene knockout technology-based Glrx1 gene knockout kit of the present invention preferably further comprises Cas9 mRNA or a Cas9 expression plasmid for expressing Cas9 mRNA.
  • the sgRNA sequence used in this experiment is highly efficient and difficult to off target.
  • the second is the optimization of the Cas9sgRNA system, which makes the mouse progeny more positive and the off-target rate low.
  • the Glrx1 knockout mice produced by this technique solve the bottleneck problem of high gene off-target rate and low survival rate of animals in traditional gene knockout technology, and can be widely used in the study of dietary nutrition and health, oxidative stress and related diseases. application.
  • the construction method of the Glrx1 gene knockout animal model based on CRISPR/Cas9 technology is realized by the following steps:
  • Step 1 Selection and design of gRNA targeting mouse Glrx1 gene
  • the Glrx-1-Cas9-KO mouse strategy was designed as shown in Figure 1. According to the strategy, design the corresponding sgRNA sequence, according to the strategy, design the corresponding sgRNA in the corresponding position of the Glrx-1 intron, order the corresponding Oligo; sgRNA sequence is as follows:
  • Step 2 sgRNA vector construction
  • the pUC57-sgRNA vector was digested with BsaI, and after 1 h of water bath at 37 ° C, 1% agarose was electrophoresed, and the digested product was recovered. The ordered sgRNA primers are then annealed. Finally, the annealing product and the recovered enzyme-cut product were connected, transformed into E. coli, and the monoclonal antibody was selected for PCR. The PCR result was positive and sent to sequencing verification, and the correct sgRNA vector was obtained. The vector map is shown in FIG. 2 .
  • Step 3 In vitro transcription of sgRNA
  • Step 4 Microinjection of fertilized eggs
  • mice On the first day, intraperitoneal injection of horse gonadotropin gonadotropin 5IU/only, human chorionic gonadotropin injection after 46-48 hours, 2 female mice after injection of human chorionic gonadotropin Put the male mouse in the cage. On the fourth morning, the plug was checked and the score of the plug was 0.5 days.
  • the selected fertilized eggs are transferred into the prepared M2 strips and arranged in a row (about 30-50 pieces). Place the syringe on the stage of the inverted microscope so that the strip of M2 droplets is oriented perpendicular to the operator, ie on the y-axis.
  • the injection tube was inserted into the cytoplasm, and the Cas9sgRNA system (sgRNA and Cas9 mRNA) was injected.
  • the Cas9 expression plasmid was cas9D10A (plasmid #42335), Addgene; and the cytoplasm was loose and the needle was quickly removed.
  • the embryos were transferred to a Petri dish containing M16 medium and placed in a 37 ° C, 5% carbon dioxide incubator for 0.5-1.0 hours.
  • the fertilized eggs were transplanted into the E0.5 day pseudopregnant recipient.
  • F0 generation mice were born about 19-21 days after transplantation.
  • the number of birth defects was 39, and the number of surviving was 38.
  • the F0 generation mice were identified by tail-cutting after 1 week of birth, and 7 positive F0 mice were obtained.
  • the coat color was black, the sex was 5 females and 2 males, and Figure 6 was F1.
  • dNTPs 0.5 10 mmol/sample Taq DNA polymerase 0.25 5 enzyme live units / microliter template 1 ⁇ 100 ng / microliter
  • Step 6 Sexual maturity and F0 mice, F1 generation mouse identification
  • F0 mice were sexually matured at 8 weeks of age and C57BL/6J mice were backcrossed.
  • the F1 mice were tail-tailed at 1 week of age, and 6 positive F1 heterozygotes were obtained. The list is as follows:
  • the F1 generation was identified from the mRNA level and enzymatic sequencing, and the mRNA level was achieved by qPCR. The conditions were the same as above. From the sequencing results, it can be seen that the length of the 61#, 62#, 64#, 73#, 74#, 75# and wild type comparison sequences is at least -7588 bp, which means that E1-E2 is deleted.
  • Example 1 differs from Example 1 in that the single-stranded DNA template and primer sequence used in step three are 2074-Glrx-gtF1.
  • the other steps were the same as in Example 1; the results were the same as in Example 1.
  • Example 2 differs from Example 1 in that the variety of the caged male in step 4 is preferably a C57BL/6J male.
  • the other steps are the same as in the first embodiment.

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Abstract

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sgRNA的选择和设计，二、sgRNA载体构建，三、sgRNA体外转录，四、小鼠受精卵注射，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。

Description

一种基于CRISPR/Cas9技术的Glrx1基因敲除动物模型的构建方法 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 guide RNA)，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 nucleases(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 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Cas9mRNA，转录好的sgRNA备用；试剂盒名称：AM1354+AM1908,Ambion by Life Technologies；
步骤四：Cas9sgRNA体系(Cas9mRNA和sgRNA)的受精卵显微注射；Cas9表达质粒为cas9D10A(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Cas9mRNA或用于表达Cas9mRNA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Cas9sgRNA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Cas9mRNA，转录好的sgRNA和Cas9mRNA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Cas9sgRNA体系(sgRNA和Cas9mRNA)，Cas9表达质粒为cas9D10A(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：
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(二价) 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
PCR检测策略如图3所示。
Figure PCTCN2018085432-appb-000001
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#：
Figure PCTCN2018085432-appb-000002
73#，74#，75#：
Figure PCTCN2018085432-appb-000003
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。

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，其引物序列如SEQ ID NO.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；
    步骤四：Cas9 mRNA和sgRNA组成的Cas9 sgRNA体系的受精卵显微注射，其中，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。
  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。
  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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