WO2020000438A1 - Method for applying crispr/cas9 system in knocking out mice ailim gene - Google Patents

Method for applying crispr/cas9 system in knocking out mice ailim gene Download PDF

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WO2020000438A1
WO2020000438A1 PCT/CN2018/093842 CN2018093842W WO2020000438A1 WO 2020000438 A1 WO2020000438 A1 WO 2020000438A1 CN 2018093842 W CN2018093842 W CN 2018093842W WO 2020000438 A1 WO2020000438 A1 WO 2020000438A1
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ailim
gene
grna
cas9
crispr
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PCT/CN2018/093842
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French (fr)
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毛吉炎
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深圳市博奥康生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system.
  • AILIM is expressed on activated T cells.
  • the interaction between AILIM and its ligand plays an important biological role in the regulation of the body's immune response. It has been confirmed that this signal significantly up-regulates the expression of IL-4, IL-5, IL-10 and IL-13. In particular, the significant up-regulation of IL-4 and IL-10 suggests that this signal is extremely important for the immune response of Th2 cells.
  • a variety of cytokines produced by T cells can be activated by AILIM.
  • AILIM The dynamic expression level of AILIM plays a key role in determining the direction of T cell polarization. It plays an important role in tumor immunotherapy. A lot of research is needed to achieve clinical transformation, but the lack of AILIM gene in the prior art has been knocked out. Mice have caused some obstacles to the progress of related research.
  • the invention provides a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system.
  • the technical solution adopted by the present invention is: a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system, which is characterized by including a mouse AILIM gene specific target site sequence, and its base The base sequence is shown in Figure 1; the gRNA oligonucleotide sequence that specifically targets the AILIM gene is shown in Figure 2.
  • An in vitro transcription vector comprising the gRNA.
  • the in vitro transcription vector of the gRNA is pMD19T-T7-gRNA
  • the in vitro transcription vector of the Cas9 protein is pST1374-NLS-flag-linker-Cas9.
  • a method for knocking out mouse AILIM gene using CRISPR / Cas9 system including the following steps:
  • the gRNA and Cas9 mRNA of the above steps are injected into the cytoplasm of mouse fertilized eggs, cultured in vitro and transplanted into the fallopian tubes of adult female mice for generating transgenic mice containing AILIM gene mutations.
  • the final concentrations are: Cas9 mRNA 30 ng / ⁇ l, and gRNA 12 ng / ⁇ l.
  • gRNA and Cas9 The mRNA is transfected into the cell at the same time, and the AILIM gene is knocked out to study the function of the AILIM gene;
  • Figure 1 is a sequence diagram of a mouse AILIM gene-specific target site
  • Figure 2 is a gRNA sequence diagram designed for targeting the AILIM gene.
  • gRNA was digested and ligated through Kpn I and Sph I sites, and inserted into pMD19T-T7-gRNA.
  • the CRISPR / Cas9 system for the AILIM gene is: in vitro transcription vectors pMD19T-T7-gRNA and pST1374-NLS-flag-linker-Cas9.
  • the constructed vector pMD19T-T7-gRNA and Cas9 mRNA in vitro transcription vector pST1374-NLS-flaglinker-Cas9 were used to perform T7 promoter-mediated in vitro transcription.
  • First pMD19T-T7-gRNA was linearized with EcoR V, and pST1374-NLS-flag-linker-Cas9 was linearized with Age I; then T7 in vitro transcription kit (Biomax) was used to perform pMD19T-T7-gRNA, pST1374- NLS-flag-linker-Cas9 in vitro transcription; MEGA Clear kit (Ambion) was used to purify gRNA and Cas9 mRNA.
  • Single-cell embryos were collected from naturally mated donor females by surgery, and the premixed gRNA and Cas9 mRNA were mixed with a microinjector (the final concentration after mixing was Cas9 mRNA 30 ng / ⁇ l, gRNA 12 ng / ⁇ l) into the cytoplasm of the fertilized eggs. Fertilized eggs transferred to Quinn ’s after injection In Advantage Cleavage Medium, the cells were cultured in vitro at 37 ° C for 24 h, and then transplanted into recipient female mice to generate transgenic mice containing AILIM gene mutations.
  • gRNA and Cas9 The mRNA is transfected into the cell at the same time, and the AILIM gene is knocked out to study the function of the AILIM gene;

Abstract

Provided is a method for applying a CRISPR/Cas9 system in knocking out mice AILIM gene.

Description

一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法Method for knocking out mouse AILIM gene using CRISPR / Cas9 system 技术领域Technical field
本发明属于动物基因工程和遗传修饰领域，特别涉及一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法。 The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system.
背景技术Background technique
AILIM在活化后的T细胞上表达。AILIM与其配体相互作用在机体的免疫反应调节中发挥重要的生物学作用,已证实该信号显著上调IL-4、IL-5、IL-10和IL-13等细胞因子的表达。尤其是对IL-4、IL-10的显著上调作用提示该信号对Th2细胞的免疫应答极其重要T细胞产生的多种细胞因子可由AILIM调节活化。AILIM is expressed on activated T cells. The interaction between AILIM and its ligand plays an important biological role in the regulation of the body's immune response. It has been confirmed that this signal significantly up-regulates the expression of IL-4, IL-5, IL-10 and IL-13. In particular, the significant up-regulation of IL-4 and IL-10 suggests that this signal is extremely important for the immune response of Th2 cells. A variety of cytokines produced by T cells can be activated by AILIM.
技术问题technical problem
AILIM的动态表达水平在决定T细胞极化方向上起关键作用，其在肿瘤的免疫治疗中起重要的作用，需做大量研究方可实现临床转化，但现有技术中缺乏AILIM基因被敲除的小鼠，对相关研究的进展造成了一定的阻碍。The dynamic expression level of AILIM plays a key role in determining the direction of T cell polarization. It plays an important role in tumor immunotherapy. A lot of research is needed to achieve clinical transformation, but the lack of AILIM gene in the prior art has been knocked out. Mice have caused some obstacles to the progress of related research.
技术解决方案Technical solutions
本发明提供了一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法。The invention provides a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system.
为实现上述目的，本发明采取的技术方案为：一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：包括针对小鼠AILIM基因特异性靶位点序列，其碱基序列如图 1所示；包括特异性靶向AILIM基因的gRNA寡核苷酸序列，其碱基序列如图 2所示。In order to achieve the above object, the technical solution adopted by the present invention is: a method for knocking out mouse AILIM gene by using CRISPR / Cas9 system, which is characterized by including a mouse AILIM gene specific target site sequence, and its base The base sequence is shown in Figure 1; the gRNA oligonucleotide sequence that specifically targets the AILIM gene is shown in Figure 2.
包括所述的gRNA的体外转录载体。An in vitro transcription vector comprising the gRNA.
所述gRNA的体外转录载体为pMD19T-T7-gRNA，Cas9蛋白的体外转录载体为pST1374-NLS-flag-linker-Cas9。The in vitro transcription vector of the gRNA is pMD19T-T7-gRNA, and the in vitro transcription vector of the Cas9 protein is pST1374-NLS-flag-linker-Cas9.
一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，包括以下步骤：A method for knocking out mouse AILIM gene using CRISPR / Cas9 system, including the following steps:
S1. 构建特异性靶向AILIM基因的gRNA的体外转录载体；通过体外转录得到靶向AILIM基因的gRNA；S1. Construct an in vitro transcription vector that specifically targets the AILIM gene gRNA; obtain gRNA that targets the AILIM gene by in vitro transcription;
S2. 体外转录Cas9蛋白的体外转录载体，得到Cas9 mRNA；S2. In vitro transcription vector for Cas9 protein in vitro, to obtain Cas9 mRNA;
S3. 将以上步骤的gRNA及Cas9 mRNA，注射入小鼠受精卵细胞质中，经体外培养后移植入成年雌性小鼠输卵管中，用于产生含AILIM基因突变的转基因小鼠。S3. The gRNA and Cas9 mRNA of the above steps are injected into the cytoplasm of mouse fertilized eggs, cultured in vitro and transplanted into the fallopian tubes of adult female mice for generating transgenic mice containing AILIM gene mutations.
在步骤S3中gRNA及Cas9 mRNA混合后，终浓度为：Cas9 mRNA 30 ng/μl，gRNA 12 ng/μl。After mixing gRNA and Cas9 mRNA in step S3, the final concentrations are: Cas9 mRNA 30 ng / μl, and gRNA 12 ng / μl.
有益效果Beneficial effect
（1）gRNA与Cas9 mRNA同时转染至细胞，敲除AILIM基因，用于研究AILIM基因的功能；(1) gRNA and Cas9 The mRNA is transfected into the cell at the same time, and the AILIM gene is knocked out to study the function of the AILIM gene;
（2）将gRNA与Cas9 mRNA同时注射入受精卵， 然后通过胚胎移植用于生产敲除AILIM的转基因小鼠；(2) Simultaneously inject gRNA and Cas9 mRNA into fertilized eggs, and then use embryo transfer to produce transgenic mice that knock out AILIM;
（3）将gRNA与Cas9 mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞，通过核移植的方法产生含AILIM基因突变的转基因小鼠。(3) Transfect gRNA and Cas9 mRNA into cells at the same time. After screening, positive cells are used as donor cells, and transgenic mice containing AILIM gene mutations are generated by nuclear transfer.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为小鼠AILIM基因特异性靶位点序列图；Figure 1 is a sequence diagram of a mouse AILIM gene-specific target site;
图2为靶向AILIM基因设计的gRNA序列图。Figure 2 is a gRNA sequence diagram designed for targeting the AILIM gene.
本发明的实施方式Embodiments of the invention
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention is described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
实施例一：靶向Example 1: Targeting AILIMAILIM 基因的genetic CRISPR/Cas9CRISPR / Cas9 系统的构建System construction
在GenBank中找到小鼠AILIM基因的序列并设计gRNA，其靶位点序列如图1所示，gRNA序列如图2所示。Find the sequence of mouse AILIM gene and design gRNA in GenBank. The target site sequence is shown in Figure 1 and the gRNA sequence is shown in Figure 2.
含有特定gRNA序列的pMD19T-T7-gRNA载体的构建：Construction of pMD19T-T7-gRNA vector containing specific gRNA sequences:
（1） 设计并合成识别AILIM基因的gRNA；(1) Design and synthesize gRNA that recognizes AILIM gene;
（2） 合成后的gRNA寡核苷酸进行体外退火；(2) annealing the synthesized gRNA oligonucleotide in vitro;
（3）将gRNA 通过Kpn I和Sph I位点进行酶切、连接，插入到pMD19T-T7-gRNA中。(3) gRNA was digested and ligated through Kpn I and Sph I sites, and inserted into pMD19T-T7-gRNA.
针对AILIM基因的CRISPR/Cas9系统即为： 体外转录载体pMD19T-T7-gRNA和pST1374-NLS-flag-linker-Cas9。The CRISPR / Cas9 system for the AILIM gene is: in vitro transcription vectors pMD19T-T7-gRNA and pST1374-NLS-flag-linker-Cas9.
实施例二：体外转录Example 2: In vitro transcription
      利用构建好的载体pMD19T-T7-gRNA和Cas9 mRNA的体外转录载体pST1374-NLS-flaglinker-Cas9进行以T7启动子介导的体外转录。首先将pMD19T-T7-gRNA用EcoR V线性化，pST1374-NLS-flag-linker-Cas9用Age I线性化；然后用T7体外转录试剂盒（百奥迈科）进行pMD19T-T7-gRNA、pST1374-NLS-flag-linker-Cas9的体外转录；最后用MEGA Clear kit（Ambion）进行gRNA、Cas9 mRNA的纯化。The constructed vector pMD19T-T7-gRNA and Cas9 mRNA in vitro transcription vector pST1374-NLS-flaglinker-Cas9 were used to perform T7 promoter-mediated in vitro transcription. First pMD19T-T7-gRNA was linearized with EcoR V, and pST1374-NLS-flag-linker-Cas9 was linearized with Age I; then T7 in vitro transcription kit (Biomax) was used to perform pMD19T-T7-gRNA, pST1374- NLS-flag-linker-Cas9 in vitro transcription; MEGA Clear kit (Ambion) was used to purify gRNA and Cas9 mRNA.
实施例三：应用靶向小鼠Example 3: Application of Targeted Mice AILIMAILIM 基因的genetic CRISPR/Cas9CRISPR / Cas9 系统产生含The system contains AILIMAILIM 基因突变的转基因小鼠Genetically modified transgenic mice
通过手术在自然交配的供体雌鼠体内收集处于单细胞阶段的胚胎， 利用显微注射仪将预混好的gRNA和Cas9 mRNA（混合后终浓度为Cas9 mRNA 30 ng/μl，gRNA 12 ng/μl）注入受精卵的细胞质中。注射后的受精卵转移至Quinn’ s Advantage Cleavage Medium中，37℃体外培养24 h，然后移植至受体雌鼠体内，用于产生含AILIM基因突变的转基因小鼠。Single-cell embryos were collected from naturally mated donor females by surgery, and the premixed gRNA and Cas9 mRNA were mixed with a microinjector (the final concentration after mixing was Cas9 mRNA 30 ng / μl, gRNA 12 ng / μl) into the cytoplasm of the fertilized eggs. Fertilized eggs transferred to Quinn ’s after injection In Advantage Cleavage Medium, the cells were cultured in vitro at 37 ° C for 24 h, and then transplanted into recipient female mice to generate transgenic mice containing AILIM gene mutations.
工业实用性Industrial applicability
（1）gRNA与Cas9 mRNA同时转染至细胞，敲除AILIM基因，用于研究AILIM基因的功能；(1) gRNA and Cas9 The mRNA is transfected into the cell at the same time, and the AILIM gene is knocked out to study the function of the AILIM gene;
（2）将gRNA与Cas9 mRNA同时注射入受精卵， 然后通过胚胎移植用于生产敲除AILIM的转基因小鼠；(2) gRNA and Cas9 mRNA is injected into fertilized eggs at the same time, and then used to produce transgenic mice with AILIM knockout through embryo transfer;
（3）将gRNA与Cas9 mRNA同时转染至细胞，筛选后用阳性细胞作为供核细胞，通过核移植的方法产生含AILIM基因突变的转基因小鼠。(3) Transfect gRNA and Cas9 mRNA into cells at the same time. After screening, positive cells are used as donor cells, and transgenic mice containing AILIM gene mutations are generated by nuclear transfer.

Claims (5)

  1. 一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：包括针对小鼠AILIM基因特异性靶位点序列，其碱基序列如图 1所示；包括特异性靶向AILIM基因的gRNA寡核苷酸序列，其碱基序列如图 2所示。A method for knocking out mouse AILIM gene using CRISPR / Cas9 system, which is characterized in that it includes a mouse AILIM gene-specific target site sequence, whose base sequence is shown in Figure 1; The nucleotide sequence of the gRNA oligonucleotide of the AILIM gene is shown in FIG. 2.
  2. 根据权利要求1所述的一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：包括所述的gRNA的体外转录载体。The method for knocking out mouse AILIM gene using the CRISPR / Cas9 system according to claim 1, characterized in that it comprises the gRNA in vitro transcription vector.
  3. 根据权利要求1所述的一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：所述gRNA的体外转录载体为pMD19T-T7-gRNA，Cas9蛋白的体外转录载体为pST1374-NLS-flag-linker-Cas9。The method for knocking out mouse AILIM gene using the CRISPR / Cas9 system according to claim 1, wherein the in vitro transcription vector of gRNA is pMD19T-T7-gRNA, and the in vitro transcription vector of Cas9 protein is pST1374-NLS-flag-linker-Cas9.
  4. 根据权利要求1所述的一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：包括以下步骤：The method for knocking out mouse AILIM gene using the CRISPR / Cas9 system according to claim 1, comprising the following steps:
    S1.构建特异性靶向AILIM基因的gRNA的体外转录载体；通过体外转录得到靶向AILIM的gRNA；S1. Construct an in vitro transcription vector that specifically targets AILIM gene gRNA; obtain gRNA that targets AILIM by in vitro transcription;
    S2.体外转录Cas9蛋白的体外转录载体，得到Cas9 mRNA；S2. In vitro transcription vector for Cas9 protein in vitro to obtain Cas9 mRNA;
    S3.将以上步骤的gRNA及Cas9 mRNA，注射入小鼠受精卵细胞质中，经体外培养后移植入成年雌性小鼠输卵管中，用于产生含AILIM基因突变的转基因小鼠。S3. The gRNA and Cas9 mRNA of the above steps are injected into the cytoplasm of mouse fertilized eggs, cultured in vitro, and transplanted into the fallopian tubes of adult female mice for generating transgenic mice containing AILIM gene mutations.
  5. 如权利要求4所述的一种应用CRISPR/Cas9系统对小鼠AILIM基因进行敲除的方法，其特征在于：在步骤S3中gRNA及Cas9 mRNA混合后，终浓度为：Cas9 mRNA 30 ng/μl，gRNA 12 ng/μl。 The method for knocking out mouse AILIM gene using the CRISPR / Cas9 system according to claim 4, characterized in that after the gRNA and Cas9 mRNA are mixed in step S3, the final concentration is: Cas9 mRNA 30 ng / μl , GRNA 12 ng / μl.
PCT/CN2018/093842 2018-06-29 2018-06-29 Method for applying crispr/cas9 system in knocking out mice ailim gene WO2020000438A1 (en)

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WO2015089473A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation

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WO2015089473A1 (en) * 2013-12-12 2015-06-18 The Broad Institute Inc. Engineering of systems, methods and optimized guide compositions with new architectures for sequence manipulation
CN104531704A (en) * 2014-12-09 2015-04-22 中国农业大学 Method for knocking off animal FGF5 gene by using CRISPR-Cas9 system

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