技术领域technical field
本发明属于动物基因工程和遗传修饰领域，具体地说，涉及一种利用CRISPR-Cas9系统敲除动物myostatin基因的方法。The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for knocking out animal myostatin gene by using a CRISPR-Cas9 system.
背景技术Background technique
自上世纪80年代基因工程兴起以来，大量基因编辑技术出现以满足科研需要，其中的基因打靶技术是一种在高等动物中对基因进行定点精细修饰的技术。传统基因打靶技术依赖体内自发的同源重组(HR,homologous recombination)，效率大约只有1/106。近年来为了解决同源重组效率低下的问题，人们通过人工构建的杂合分子对特定的DNA序列进行切割，以此来提高基因打靶的效率，其中以核酸内切酶为核心的人工复合分子最受关注。Since the rise of genetic engineering in the 1980s, a large number of gene editing technologies have emerged to meet the needs of scientific research. Among them, gene targeting technology is a technology for targeted and fine modification of genes in higher animals. Traditional gene targeting technology relies on spontaneous homologous recombination (HR, homologous recombination) in vivo, and the efficiency is only about 1/106 . In recent years, in order to solve the problem of low efficiency of homologous recombination, people use artificially constructed hybrid molecules to cut specific DNA sequences to improve the efficiency of gene targeting. Among them, artificial compound molecules with endonuclease as the core are the most attention.
Myostatin基因(GDF8)是转移生长因子超家族的成员，它在动物体内作为肌肉生长的负调节因子，在肌肉的形成及分化发育过程中发挥重要作用。自然界中天然存在myostatin基因的自然突变型动物，如比利时蓝牛、皮尔蒙特牛等，它们都具有肌肉极端发达的表型，因此在生产领域具有极为重要的实用价值。而通过传统的基因工程技术对哺乳动物，尤其是大动物进行myostatin基因进行敲除存在很大的技术难度，因而一直以来大动物的myostatin基因敲除效率非常低下。Myostatin gene (GDF8) is a member of the transforming growth factor superfamily. It acts as a negative regulator of muscle growth in animals and plays an important role in the formation, differentiation and development of muscle. Natural mutant animals with myostatin gene naturally exist in nature, such as Belgian blue cattle, Pyrmont cattle, etc., all of which have extremely muscular phenotypes, so they have extremely important practical value in the field of production. However, it is very difficult to knock out the myostatin gene in mammals, especially large animals, through traditional genetic engineering techniques, so the efficiency of myostatin gene knockout in large animals has always been very low.
CRISPR/Cas9系统包括一个具备DNA结合和切割的Cas9蛋白以及负责特异性识别DNA序列并引导Cas9蛋白特异性结合到目的DNA位点的sgRNA。在动物体内，Cas9蛋白与sgRNA首先结合成一个蛋白复合体，然后通过sgRNA的特异性识别作用，在基因组中识别到特定的目标DNA序列并一到蛋白复合体结合到DNA链上。然后通过Cas9的核酸内切酶活性在目标位点将DNA切开，形成一个DNA双链断裂(DSB)。通过诱导自体的DNA修复机制发挥作用，如同源重组或非同源末端连接(NHEJ，Non-homologous end joining)，从而在该位点产生基因突变，从而到达基因打靶的目的。The CRISPR/Cas9 system includes a Cas9 protein capable of DNA binding and cutting and sgRNA responsible for specifically recognizing DNA sequences and guiding the Cas9 protein to specifically bind to the target DNA site. In animals, Cas9 protein and sgRNA first combine to form a protein complex, and then through the specific recognition of sgRNA, a specific target DNA sequence is recognized in the genome and the protein complex binds to the DNA chain. The DNA is then cut at the target site by the endonuclease activity of Cas9, forming a DNA double-strand break (DSB). By inducing autologous DNA repair mechanisms, such as homologous recombination or non-homologous end joining (NHEJ, Non-homologous end joining), gene mutations are generated at this site, thereby achieving the purpose of gene targeting.
与传统的同源重组介导的基因打靶相比较，利用CRISPR/Cas9系统对哺乳动物进行基因打靶具有操作简便，效率高，适用物种广泛等优点，尤其可以一次性的实现多基因打靶，这在动物遗传修饰及疾病模型研究中都有极其广阔的应用前景。Compared with the traditional gene targeting mediated by homologous recombination, the use of CRISPR/Cas9 system for gene targeting in mammals has the advantages of simple operation, high efficiency, and wide range of applicable species. In particular, it can realize multi-gene targeting at one time. It has extremely broad application prospects in animal genetic modification and disease model research.
传统的基因打靶技术依赖生物体内自发的同源重组现象，因而效率很低，随着技术发展后来便出现了锌指核酸酶(ZFNs，zinc fingernucleases)和转录激活子样效应子介导核酸酶(TALENs，transcriptionactivator-like effector nucleases)。这两种系统结构比较相似，每个蛋白分子都是由FolkI核酸内切酶结构域和DNA识别结构域两部分构成，通过每个蛋白分子中的特异性DNA识别域识别基因组中的特定目的序列，从而将FolkI内切酶定位至靶位点。由于FolkI核酸内切酶需要在DNA双链上形成二聚体形式才能发挥核酸内切酶的功能，因而ZFNs和TALENs在基因打靶时都需要两个分子分别定位在靶位点的两条DNA链上，才可以发挥核酸内切酶的作用，对DNA进行切割产生双链断裂(DSB).Traditional gene targeting technology relies on the phenomenon of spontaneous homologous recombination in organisms, so the efficiency is very low. With the development of technology, zinc finger nucleases (ZFNs, zinc fingernucleases) and transcription activator-like effector-mediated nucleases ( TALENs, transcription activator-like effector nucleases). The structures of these two systems are relatively similar. Each protein molecule is composed of two parts, the FolkI endonuclease domain and the DNA recognition domain. The specific target sequence in the genome is recognized by the specific DNA recognition domain in each protein molecule. , thereby positioning the FolkI endonuclease to the target site. Since the FolkI endonuclease needs to form a dimer on the double strand of DNA to function as an endonuclease, both ZFNs and TALENs need two DNA strands that are respectively positioned at the target site during gene targeting. In order to play the role of endonuclease, DNA can be cut to produce double-strand breaks (DSB).
虽然ZFNs和TALENs相比于传统的同源重组具有打靶效率高的优点，但是它们依然存在很多的缺陷，主要包括：1、ZFNs和TALENs的DNA切割结构域均为FolkI，它必须以二聚体形式发挥作用，因而对哺乳动物进行基因打靶时至少要采用两个DNA表达结构，这在细胞转染时会对转染效率有更高的要求；2、ZFNs的设计和生产相对复杂，成本较高，应用于大量的哺乳动物基因打靶时成本难以控制在可以承受的范围；3、ZFNs和TALENs的DNA识别规则和设计要求较为严格，可能出现靶基因序列中无法找到合适的ZFNs、TALENs识别区，从而无法应用其进行基因打靶的情况；4、针对不同基因或者不同物种的同一基因进行基因打靶时，都需要从新设计和构建新的ZFNs、TALENs表达质粒或mRNA，操作繁复；5、ZFNs和TALENs在进行多基因打靶时，受载体和mRNA分子大小的限制，很难获得较高的打靶效率。Although ZFNs and TALENs have the advantages of high targeting efficiency compared with traditional homologous recombination, they still have many defects, mainly including: 1. The DNA cutting domain of ZFNs and TALENs is FolkI, which must form a dimer Therefore, at least two DNA expression structures must be used for gene targeting in mammals, which will have higher requirements for transfection efficiency during cell transfection; 2. The design and production of ZFNs are relatively complicated and costly High, and it is difficult to control the cost within an acceptable range when applied to a large number of mammalian gene targeting; 3. The DNA recognition rules and design requirements of ZFNs and TALENs are relatively strict, and it may not be possible to find suitable ZFNs and TALENs recognition regions in the target gene sequence , so that it cannot be used for gene targeting; 4. When targeting different genes or the same gene of different species, it is necessary to design and construct new ZFNs and TALENs expression plasmids or mRNAs, and the operation is complicated; 5. ZFNs and When TALENs perform multi-gene targeting, it is difficult to obtain high targeting efficiency due to the limitation of the size of the carrier and mRNA molecules.
发明内容Contents of the invention
本发明的目的是提供一种CRISPR-Cas9系统敲除动物myostatin基因的方法。The purpose of the present invention is to provide a method for knocking out animal myostatin gene by CRISPR-Cas9 system.
本发明的另一个目的是提供特异性靶向myostatin基因的sgRNA。Another object of the present invention is to provide sgRNA specifically targeting myostatin gene.
本发明首先比对了不同物种(人类，小鼠，猪，牛，绵羊，山羊)的myostatin基因序列，从中找到了一个相对保守的区域，在这个区域中进行sgRNA的设计并获得了一条sgRNA的序列信息。该特异性靶向myostatin基因第二外显子的sgRNA，其DNA序列如SEQ ID NO.1所示。本发明提供了该特异性靶向myostatin基因第二外显子的sgRNA在敲除动物myostatin基因中的应用。The present invention first compared the myostatin gene sequences of different species (human, mouse, pig, cow, sheep, goat), found a relatively conserved region, designed sgRNA in this region and obtained a sgRNA sequence information. The sgRNA specifically targeting the second exon of the myostatin gene has a DNA sequence as shown in SEQ ID NO.1. The present invention provides the application of the sgRNA specifically targeting the second exon of myostatin gene in knocking out animal myostatin gene.
本发明还提供了含有上述sgRNA的DNA序列的载体。The present invention also provides a vector containing the DNA sequence of the above sgRNA.
在本发明的实施例中，提供的上述载体为pX330-M2。In an embodiment of the present invention, the above-mentioned vector provided is pX330-M2.
具体地，pX330-M2的构建方法为：(1)设计并合成识别myostatin第二外显子的sgRNA识别区DNA序列，如SEQ ID NO.1所示；(2)合成后的sgRNA序列进行磷酸化后梯度降温退火，具体步骤为将合成的oligo DNA与10X T4Ligation Buffer和T4PNK以2:2:1的比例混合后再加入3倍体积的水补齐体系，然后37℃孵育30min，再95℃5min变性，之后在以5℃每分钟的速率降温至25℃完成反应以产生磷酸化粘性末端，同时BbsI酶切载体pX330产生粘性末端；(3)以T4连接酶将这个片段与pX330进行连接，获得真核CRISPR-Cas9系统载体pX330-M2。Specifically, the construction method of pX330-M2 is: (1) design and synthesize the DNA sequence of the sgRNA recognition region that recognizes the second exon of myostatin, as shown in SEQ ID NO.1; (2) phosphorylate the synthesized sgRNA sequence Gradient cooling and annealing after melting, the specific steps are to mix the synthesized oligo DNA with 10X T4Ligation Buffer and T4PNK at a ratio of 2:2:1, then add 3 times the volume of water to make up the system, then incubate at 37°C for 30min, then incubate at 95°C Denatured for 5 minutes, then cooled to 25°C at a rate of 5°C per minute to complete the reaction to generate phosphorylated cohesive ends, while BbsI digested vector pX330 to generate sticky ends; (3) Ligated this fragment with pX330 with T4 ligase, The eukaryotic CRISPR-Cas9 system vector pX330-M2 was obtained.
本发明还提供了体外转录载体pIVT-M2-T载体。The invention also provides the in vitro transcription vector pIVT-M2-T vector.
所述体外转录载体pIVT-M2-T的构建方法为：设计含有T7启动子的上游引物，序列如SEQ ID NO.2所示和与其匹配的下游引物，序列如SEQ ID NO.3所示，以载体pX330-M2为模板PCR扩增获得可以用于体外转录的IVT-M2片段，再以TA克隆的方式将该片段插入pMD18-T载体中，获得用于体外转录的载体pIVT-M2-T。The construction method of the in vitro transcription vector pIVT-M2-T is: design an upstream primer containing a T7 promoter, the sequence of which is shown in SEQ ID NO.2 and a matching downstream primer, the sequence of which is shown in SEQ ID NO.3, Using the vector pX330-M2 as a template, the IVT-M2 fragment that can be used for in vitro transcription was obtained by PCR amplification, and then inserted into the pMD18-T vector by TA cloning to obtain the vector pIVT-M2-T for in vitro transcription .
本发明提供了用于敲除动物myostatin基因的CRISPR-Cas9系统，含有特异性靶向myostatin基因第二外显子的sgRNA的表达载体和Cas9蛋白的体外转录载体。The present invention provides a CRISPR-Cas9 system for knocking out animal myostatin gene, which contains an expression vector of sgRNA specifically targeting the second exon of myostatin gene and an in vitro transcription vector of Cas9 protein.
本发明的实施例中，所述含有特异性靶向myostatin基因第二外显子的sgRNA的表达载体为pIVT-M2-T，Cas9蛋白的体外转录载体为pCas9-puro3。In an embodiment of the present invention, the expression vector containing the sgRNA specifically targeting the second exon of the myostatin gene is pIVT-M2-T, and the in vitro transcription vector of the Cas9 protein is pCas9-puro3.
其中，pCas9-puro3是通过以下方法构建得到的：(1)以内切酶AgeI和NotI酶切载体pX330-U6-Chimeric_BB-CBh-hSpCas9获得pX330载体中的Cas9表达区段；(2)以AgeI和NotI线性化载体pIRES-puro3；(3)将步骤(1)的Cas9表达区段与步骤(2)的线性化载体pIRES-puro3进行连接获得终载体pCas9-puro3。Among them, pCas9-puro3 is constructed by the following methods: (1) digest the vector pX330-U6-Chimeric_BB-CBh-hSpCas9 with endonucleases AgeI and NotI to obtain the Cas9 expression segment in the pX330 vector; (2) use AgeI and NotI NotI linearized vector pIRES-puro3; (3) connecting the Cas9 expression segment in step (1) with the linearized vector pIRES-puro3 in step (2) to obtain the final vector pCas9-puro3.
本发明提供了利用CRISPR-Cas9系统敲除动物myostatin基因的方法，包括以下步骤：The invention provides a method for knocking out the animal myostatin gene using the CRISPR-Cas9 system, comprising the following steps:
(1)构建特异性靶向myostatin基因第二外显子的sgRNA的表达载体；通过体外转录表达得到myostatin基因第二外显子sgRNA；(1) Construct an expression vector of sgRNA specifically targeting the second exon of myostatin gene; obtain the second exon sgRNA of myostatin gene through in vitro transcription and expression;
(2)构建Cas9蛋白的体外转录载体，得到Cas9mRNA；(2) Construct the in vitro transcription vector of Cas9 protein to obtain Cas9 mRNA;
(3)将步骤(1)的sgRNA和步骤(2)的Cas9mRNA纯化后，混合，注射入动物受精卵细胞质或细胞核中，经体外短时培养后移植入同种雌性动物输卵管中，或体外培养至囊胚期再移植到同种雌性动物子宫中，以生产敲除myostatin基因的动物。所述短时培养是指30min-48h。(3) After the sgRNA in step (1) and the Cas9mRNA in step (2) are purified, they are mixed, injected into the cytoplasm or nucleus of fertilized animal eggs, and transplanted into the oviduct of the same female animal after short-term culture in vitro, or cultured in vitro At the blastocyst stage, they were transplanted into the uterus of female animals of the same species to produce animals whose myostatin gene was knocked out. The short-term cultivation refers to 30min-48h.
本发明方法中，步骤(1)特异性靶向myostatin基因第二外显子的sgRNA的表达载体为pIVT-M2-T。步骤(2)的Cas9蛋白的体外转录载体为pCas9-puro3。In the method of the present invention, the expression vector of the sgRNA specifically targeting the second exon of the myostatin gene in step (1) is pIVT-M2-T. The in vitro transcription vector of the Cas9 protein in step (2) is pCas9-puro3.
步骤(1)和(2)转录产生的mRNA进行吸附柱纯化，将纯化后的mRNA利用分光光度计测定浓度。在本发明的实施例中获得的sgRNA浓度为103ng/μl，Cas9mRNA的浓度为775ng/μl，并按照,使混合后sgRNA和Cas9mRNA终浓度分别为20ng/μl和150ng/μl。The mRNA produced by transcription in steps (1) and (2) is purified by an adsorption column, and the concentration of the purified mRNA is measured by a spectrophotometer. The sgRNA concentration obtained in the embodiment of the present invention is 103ng/μl, the concentration of Cas9mRNA is 775ng/μl, and according to the following, the final concentrations of sgRNA and Cas9mRNA after mixing are 20ng/μl and 150ng/μl respectively.
步骤(3)中，sgRNA与Cas9mRNA混合前，sgRNA的浓度范围为100～200ng/μl，Cas9mRNA的浓度范围为500～2000ng/μl，sgRNA和Cas9mRNA混合后的质量比为1:10～1:2混合。In step (3), before mixing sgRNA and Cas9mRNA, the concentration range of sgRNA is 100-200ng/μl, the concentration range of Cas9mRNA is 500-2000ng/μl, and the mass ratio of sgRNA and Cas9mRNA after mixing is 1:10-1:2 mix.
优选地，二者混合后的质量比为1:7.5。Preferably, the mixed mass ratio of the two is 1:7.5.
本发明还提供了上述方法在制备敲除myostatin基因的动物中的应用。The present invention also provides the application of the above method in the preparation of animals knocking out the myostatin gene.
本发明利用CRISPR-Cas9系统进行哺乳动物基因打靶，其优点是：1、sgRNA特异性识别DNA序列中的NGG三个碱基对，识别规则简单且易分析，在靶基因中可以同时找到多个sgRNA识别位点，从而可以根据打靶要求进行选择，适用性广泛；2、相比于ZFNs和TALENs的复杂表达结构，CRISPR-Cas9系统中的Cas9表达结构是固定不变的，针对不同基因只需要将23bp的识别序列插入sgRNA表达结构中即可完成系统组建，操作简单，成本低，适用于大规模的哺乳动物基因打靶工作；3、利用CRISPR-Cas9系统继续细胞转染时，由于sgRNA表达结构很短，所以可以大大提高转染效率，也可以将Cas9和sgRNA表达结构整合到一个载体中，进一步提高转染效率，这都是ZFNs和TALENs很难做到的；4、针对同一家族的不同基因或不同物种的同一基因进行基因打靶时，可以选择基因中的保守区进行sgRNA设计，从而实现同一条sgRNA对多个基因的打靶或修饰，相比其他技术更加简便、高效；5、可以通过向哺乳动物中同时导入一个Cas9表达结构和多个sgRNA的方式轻松实现多基因同时打靶，无论从打靶效率还是操作简便程度上都是其他技术无法比拟的。The present invention uses the CRISPR-Cas9 system to target mammalian genes, and its advantages are: 1. sgRNA specifically recognizes the three base pairs of NGG in the DNA sequence, the recognition rules are simple and easy to analyze, and multiple genes can be found in the target gene sgRNA recognition site, so that it can be selected according to the targeting requirements, and has wide applicability; 2. Compared with the complex expression structure of ZFNs and TALENs, the expression structure of Cas9 in the CRISPR-Cas9 system is fixed. For different genes, only need The system can be established by inserting the 23bp recognition sequence into the sgRNA expression structure, which is easy to operate and low in cost, and is suitable for large-scale mammalian gene targeting work; 3. When using the CRISPR-Cas9 system to continue cell transfection, due to the sgRNA expression structure It is very short, so the transfection efficiency can be greatly improved, and the expression structure of Cas9 and sgRNA can be integrated into one vector to further improve the transfection efficiency, which is difficult for ZFNs and TALENs; 4. Targeting different genes of the same family When targeting a gene or the same gene of different species, the conserved region in the gene can be selected for sgRNA design, so as to realize the targeting or modification of multiple genes by the same sgRNA, which is more convenient and efficient than other technologies; 5. Through Simultaneously introducing a Cas9 expression structure and multiple sgRNAs into mammals can easily achieve simultaneous multi-gene targeting, which is unmatched by other technologies in terms of targeting efficiency and ease of operation.
本发明通过构建特异性靶向myostatin基因第二外显子的sgRNA的表达载体和Cas9蛋白的体外转录载体，获得了靶向myostatin基因第二外显子的sgRNA和Cas9蛋白的mRNA，将其混合后注入动物受精卵细胞质或细胞核中，经体外短时培养或培养数天后移植到同种雌性动物输卵管或子宫中，能够生产得到敲除myostatin基因的动物，可提高基因打靶的效率和适应性。本发明的另一个突出优点在于，本发明提供的靶向myostatin基因第二外显子的sgRNA是对比人类，小鼠，猪，牛，绵羊，山羊等多个物种的保守序列获得的，因此本发明的sgRNA可通用于多个物种的myostatin基因敲除。The present invention obtains the mRNA of the sgRNA targeting the second exon of the myostatin gene and the mRNA of the Cas9 protein by constructing the expression vector of the sgRNA specifically targeting the second exon of the myostatin gene and the in vitro transcription vector of the Cas9 protein, and mixing them After injecting into the cytoplasm or nucleus of animal fertilized eggs, after a short period of in vitro culture or culture for several days, it is transplanted into the oviduct or uterus of the same female animal to produce animals with knockout myostatin gene, which can improve the efficiency and adaptability of gene targeting. Another outstanding advantage of the present invention is that the sgRNA targeting the second exon of the myostatin gene provided by the present invention is obtained by comparing the conserved sequences of multiple species such as humans, mice, pigs, cattle, sheep, and goats. The invented sgRNA can be used for knockout of myostatin gene in multiple species.
具体实施方式Detailed ways
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.
以下实施例中涉及的试验材料和试剂：pX330-U6-Chimeric_BB-CBh-hSpCas9购自addgene，pMD18-T购自takara公司，pIRES-puro3购自Clontech公司。B6D2F1小鼠为C57BL/6和DBA/2小鼠杂交1代鼠，C57BL/6和DBA/2均购自北京维通利华实验动物技术有限公司。绵羊购自北京顺义金鑫现代农业发展有限公司。The test materials and reagents involved in the following examples: pX330-U6-Chimeric_BB-CBh-hSpCas9 was purchased from addgene, pMD18-T was purchased from takara company, and pIRES-puro3 was purchased from Clontech company. B6D2F1 mice are the first-generation mice crossed between C57BL/6 and DBA/2 mice, and both C57BL/6 and DBA/2 were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Sheep were purchased from Beijing Shunyi Jinxin Modern Agriculture Development Co., Ltd.
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中描述的具体方法进行，或者按照试剂盒和产品说明书进行。The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods described in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or carried out according to the kit and product instructions .
实施例1针对myostatin基因的CRISPR-Cas9系统的构建Example 1 Construction of the CRISPR-Cas9 system for the myostatin gene
1、比对了不同物种(人类，小鼠，猪，牛，绵羊，山羊)的myostatin基因序列，从中找到了一个相对保守的区域，在这个区域中进行sgRNA的设计并获得了一条sgRNA的序列信息。其中特异性靶向myostatin基因第二外显子的sgRNA，其DNA序列如SEQ ID NO.1所示。1. Compared the myostatin gene sequences of different species (human, mouse, pig, cow, sheep, goat), found a relatively conserved region, designed sgRNA in this region and obtained a sgRNA sequence information. The DNA sequence of the sgRNA specifically targeting the second exon of the myostatin gene is shown in SEQ ID NO.1.
2、pX330-M2的构建：(1)设计并合成识别myostatin第二外显子的sgRNA识别区DNA序列，如SEQ ID NO.1所示；(2)合成后的sgRNA序列进行磷酸化后梯度降温退火，具体步骤为将合成的oligo DNA与10X T4Ligation Buffer和T4PNK以2:2:1的比例混合后再加入3倍体积的水补齐体系，然后37℃孵育30min，再95℃，5min变性，之后在以5℃每分钟的速率降温至25℃完成反应以产生磷酸化粘性末端，同时BbsI酶切载体pX330产生粘性末端；(3)以T4连接酶分别将这两个片段与pX330进行连接，获得真核CRISPR-Cas9系统载体pX330-M2。2. Construction of pX330-M2: (1) Design and synthesize the DNA sequence of the sgRNA recognition region that recognizes the second exon of myostatin, as shown in SEQ ID NO.1; (2) Gradient after phosphorylation of the synthesized sgRNA sequence Cooling and annealing, the specific steps are to mix the synthesized oligo DNA with 10X T4Ligation Buffer and T4PNK at a ratio of 2:2:1, then add 3 times the volume of water to complete the system, then incubate at 37°C for 30min, then denature at 95°C for 5min , then cool down to 25°C at a rate of 5°C per minute to complete the reaction to generate phosphorylated cohesive ends, and at the same time, BbsI digests the vector pX330 to generate sticky ends; (3) Ligate the two fragments with pX330 with T4 ligase , to obtain the eukaryotic CRISPR-Cas9 system vector pX330-M2.
3、转录载体pIVT-M2-T的构建：设计含有T7启动子的上游引物，序列如SEQ ID NO.2所示和与其匹配的下游引物，序列如SEQ IDNO.3所示，以载体pX330-M2为模板PCR扩增获得可以用于体外转录的IVT-M2片段，再以TA克隆的方式将该片段插入pMD18-T载体中，获得用于体外转录的载体pIVT-M2-T。3. Construction of the transcription vector pIVT-M2-T: design the upstream primer containing the T7 promoter, whose sequence is shown in SEQ ID NO. M2 was used as a template to amplify the IVT-M2 fragment that can be used for in vitro transcription, and then the fragment was inserted into the pMD18-T vector by TA cloning to obtain the vector pIVT-M2-T for in vitro transcription.
4、转录载体pCas9-puro3是通过以下方法构建得到的：(1)以内切酶AgeI和NotI酶切载体pX330-U6-Chimeric_BB-CBh-hSpCas9获得pX330载体中的Cas9表达区段；(2)以AgeI和NotI线性化载体pIRES-puro3；(3)将步骤(1)的Cas9表达区段与步骤(2)的线性化载体pIRES-puro3进行连接获得终载体pCas9-puro3。4. The transcription vector pCas9-puro3 was constructed by the following methods: (1) digest the vector pX330-U6-Chimeric_BB-CBh-hSpCas9 with endonucleases AgeI and NotI to obtain the Cas9 expression segment in the pX330 vector; (2) use AgeI and NotI linearized vector pIRES-puro3; (3) connecting the Cas9 expression segment in step (1) with the linearized vector pIRES-puro3 in step (2) to obtain the final vector pCas9-puro3.
针对myostatin基因的CRISPR-Cas9系统即为：转录载体pIVT-M2-T和转录载体pCas9-puro3。The CRISPR-Cas9 system for myostatin gene is: transcription vector pIVT-M2-T and transcription vector pCas9-puro3.
实施例2体外转录Example 2 In vitro transcription
利用构建的体外转录载体pIVT-M2-T和pCas9-puro3进行T7启动子介导的体外转录，即以T7启动子作为体外转录的启动子，利用RNA聚合酶在体外实现从DNA到mRNA的转录过程，具体方法为：分别以SalI和NotI线性化载体pIVT-M2-T和pCas9-puro3，然后以线性化的体外转录载体为模板，加入T7转录酶、buffer以及rNTPs，37℃孵育6h，然后加入DNA酶37℃，15min消化去除模板DNA，再以酚仿抽提去除蛋白杂质后，乙醇沉淀获得转录后的mRNA，将转录产生的mRNA进行吸附柱纯化，具体方法为：向mRNA加入3.5倍体积的结合缓冲液和2.5倍体积的无水乙醇，混匀后加入吸附柱中，12000rpm离心10min，再以洗涤液洗涤吸附柱两次，最后用100μl的纯水溶解mRNA，将纯化后的mRNA利用分光光度计测定浓度，sgRNA浓度为103ng/μl，Cas9mRNA的浓度为775ng/μl。Use the constructed in vitro transcription vectors pIVT-M2-T and pCas9-puro3 for T7 promoter-mediated in vitro transcription, that is, use the T7 promoter as the promoter for in vitro transcription, and use RNA polymerase to achieve transcription from DNA to mRNA in vitro The process, the specific method is: use SalI and NotI to linearize the vectors pIVT-M2-T and pCas9-puro3 respectively, then use the linearized in vitro transcription vector as a template, add T7 transcriptase, buffer and rNTPs, incubate at 37°C for 6h, and then Add DNase at 37°C for 15 minutes to digest and remove template DNA, then extract protein impurities with phenolform, and then ethanol precipitate to obtain transcribed mRNA, and purify the transcribed mRNA by adsorption column. The specific method is: add 3.5 times volume of binding buffer and 2.5 times the volume of absolute ethanol, mix well and add to the adsorption column, centrifuge at 12000rpm for 10min, then wash the adsorption column twice with washing solution, and finally dissolve the mRNA with 100μl of pure water, and the purified mRNA The concentration was measured by a spectrophotometer, the sgRNA concentration was 103ng/μl, and the Cas9mRNA concentration was 775ng/μl.
实施例3利用针对myostatin基因的CRISPR-Cas9系统mRNA生产基因打靶小鼠Example 3 Production of gene-targeted mice using the CRISPR-Cas9 system mRNA targeting the myostatin gene
1、原核注射及胚胎移植1. Pronuclear injection and embryo transfer
取B6D2F1小鼠的原核期受精卵，利用显微注射仪将预混好的Cas9mRNA/sgRNA混合物(Cas9mRNA终浓度为150ng/μl，sgRNA终浓度为20ng/μl)，注射至小鼠受精卵细胞质或细胞核中。注射后的受精卵转移至培养液中短暂培养，然后移植至受体母鼠的输卵管中，生产基因打靶小鼠。The pronuclear fertilized eggs of B6D2F1 mice were taken, and the premixed Cas9mRNA/sgRNA mixture (the final concentration of Cas9mRNA was 150ng/μl, and the final concentration of sgRNA was 20ng/μl) was injected into the cytoplasm or in the nucleus. After the injection, the fertilized eggs are transferred to the culture medium for short-term culture, and then transplanted into the oviduct of recipient mother mice to produce gene-targeted mice.
2、基因打靶小鼠的鉴定2. Identification of gene targeting mice
代孕母鼠生产后，待仔鼠长至2周龄剪取1cm左右鼠尾，蛋白酶K，55℃消化后酚仿抽提提取鼠尾基因组。以鼠尾基因组为模板，设计针对myostatin第二外显子的引物，序列如Seq No.4～5进行扩增，对获得的PCR产物进行测序，如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后TA克隆至T载体中，转化后挑取阳性克隆再次进行测序，如测序结果中myostatin基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变，则可判断为myostatin基因敲除。After the birth of the surrogate mother mouse, cut off about 1 cm of mouse tail when the offspring grow to 2 weeks old, digest with proteinase K at 55°C, and extract the mouse tail genome by phenolform extraction. Using the mouse tail genome as a template, design primers targeting the second exon of myostatin, amplify the sequence such as Seq No. 4-5, and sequence the obtained PCR products. If there are double peaks near the target site in the sequencing results , it may be a successful target. Select the bimodal sample for PCR again. After the product is recovered from the gel, it is cloned into the T vector with TA. After transformation, the positive clone is picked and sequenced again. Frameshift mutation can be judged as myostatin gene knockout.
实施例4利用针对myostatin基因的CRISPR-Cas9系统mRNA进行绵羊胚胎基因打靶Example 4 Using the CRISPR-Cas9 system mRNA for the myostatin gene to perform gene targeting in sheep embryos
1、原核注射及体外培养1. Pronuclear injection and in vitro culture
从绵羊卵巢中采集卵子进行体外成熟，选取成熟的卵子进行体外受精，在原核期利用显微注射仪将预混好的Cas9mRNA/sgRNA混合物(Cas9mRNA终浓度为150ng/μl，sgRNA终浓度为20ng/μl)，注射至受精卵细胞核中。注射后的受精卵转移至培养液中继续培养。待注射后的受精卵培养至囊胚期后，取胚胎加裂解液进行裂解处理，以进行下一步的鉴定实验。Oocytes were collected from sheep ovaries for in vitro maturation, mature eggs were selected for in vitro fertilization, and the premixed Cas9mRNA/sgRNA mixture (the final concentration of Cas9mRNA was 150ng/μl, the final concentration of sgRNA was 20ng/μl) was injected using a microinjector in the pronuclear stage. μl), injected into the nucleus of fertilized eggs. After the injection, the fertilized eggs were transferred to the culture medium to continue culturing. After the injected fertilized eggs are cultured to the blastocyst stage, the embryos are taken and lysed with lysate for lysing treatment, so as to carry out the next identification experiment.
2、基因打靶结果的检测2. Detection of gene targeting results
以发育后的绵羊胚胎为模板进行PCR，引物针对myostatin的第二外显子，引物序列如Seq No.6～7，PCR产物进行测序，如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后TA克隆至T载体中，转化后挑取阳性克隆再次进行测序，如测序结果中myostatin基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变，则可判断为基因敲除，即为基因打靶阳性结果。Use the developed sheep embryo as a template to carry out PCR, and the primers are aimed at the second exon of myostatin. The primer sequence is Seq No.6~7, and the PCR products are sequenced. If there are double peaks near the target site in the sequencing results, then Possibly a target success. Select the bimodal sample for PCR again. After the product is recovered from the gel, it is cloned into the T vector with TA. After transformation, the positive clone is picked and sequenced again. A frameshift mutation can be judged as a gene knockout, which is a positive result of gene targeting.
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
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| CN201410751563.4ACN104531705A (en) | 2014-12-09 | 2014-12-09 | Method for knocking off animal myostatin gene by using CRISPR-Cas9 system |
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| CN201410751563.4ACN104531705A (en) | 2014-12-09 | 2014-12-09 | Method for knocking off animal myostatin gene by using CRISPR-Cas9 system |
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| Date | Code | Title | Description |
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| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication | Application publication date:20150422 |