WO2019237365A1 - CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA - Google Patents

CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA Download PDF

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WO2019237365A1
WO2019237365A1 PCT/CN2018/091678 CN2018091678W WO2019237365A1 WO 2019237365 A1 WO2019237365 A1 WO 2019237365A1 CN 2018091678 W CN2018091678 W CN 2018091678W WO 2019237365 A1 WO2019237365 A1 WO 2019237365A1
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mer6
gene
sgrna
crispr
cas9
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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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    • 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
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    • 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
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

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  • the invention belongs to the technical field of gene editing, and particularly relates to a method for specifically knocking out the human MER6 gene by CRISPR-Cas9 and a specific sgRNA thereof.
  • MER6 is a member of the immunoglobulin superfamily, which is widely expressed on the surface of cells. It can interact with signal-regulating protein ⁇ , thrombospondin, and integrin to mediate a series of reactions such as apoptosis, proliferation, and immunity.
  • MER6 is a protective receptor for cancer cells from being attacked by the host's immune system. Studies have shown that it plays an important role in tumor immunotherapy. A lot of research is needed to achieve clinical transformation, but the existing technology lacks the promotion of the MER6 gene. Expressed plasmids have hindered the progress of related research.
  • the purpose of the present invention is to provide a method for specifically knocking out the human MER6 gene by using CRISPR-Cas9, which has a simple structure, a reasonable design, and convenient use, and its specific sgRNA. And designed and synthesized a set of sgRNAs that specifically targeted the MER6 gene by using CRISPR-Cas9 to specifically knock out the MER6 gene, and ligated the sgRNA with a linear PX330 plasmid into a vector, and transfected the cells to achieve the MER6 gene Knockout can achieve a permanent effect; it provides highly efficient sgRNA; sgRNA only needs to synthesize a small amount of polynucleotide fragments, which can be produced in large quantities.
  • the method for specifically knocking out the human MER6 gene and its specific sgRNA according to the present invention adopt the following technical scheme:
  • the selected sgRNA add CCGG to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain the complementary strand of its corresponding DNA, and add AAAC to its 5' end to obtain a reverse oligonucleotide .
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse sequence oligonucleotide sequences of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, the PX330 vector could be ligated. Double-stranded annealing product.
  • the invention also provides an sgRNA that specifically targets the MER6 gene, the sequence of which is shown in SEQ ID NO. 1.
  • the present invention has the following advantages and effects:
  • the function of the antibody is only a temporary blocking effect.
  • the present invention can both knock out multiple coding sequences of MER6;
  • Antibodies can only target extracellular targets, and the present invention can target both extracellular and intracellular targets;
  • Figure 1 is the structure of the PX330 carrier
  • Figure 2 is a T7EN1 digestion identification of sgRNA / Cas9-mediated gene human MER6 specific cleavage, in which 1- untreated control group 293T cells, 2- experimental group 293T cells transfected with PX330-MER6 vector.
  • the selected sgRNA sequence (SEQ ID No. 1), add CACC to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain its complementary strand, and add AAAC to its 5' end to obtain a reverse oligonucleotide.
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse oligonucleotides of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, a double pair that can be ligated into the PX330 vector was formed. Stranded sgRNA oligonucleotide.
  • the denaturing and annealing system is:
  • Example 2 Utilization CRISPR-Cas9 Specific knockout MER6 gene
  • the enzyme digestion system and conditions are as follows:
  • the double-stranded sgRNA oligonucleotide that can be ligated into the PX330 vector obtained after denaturation and annealing is ligated with the linearized PX330 plasmid to obtain the PX330-MER6 plasmid.
  • connection system is as follows:
  • the high-fidelity PCR enzyme was amplified, and the PCR cleanup was purified to obtain the PCR recovered product.
  • 100 ng was uniformly diluted to 20 ⁇ L for denaturation and annealing. The procedures were: 95 ° C, 3min; 95-85 ° C, cooling at 2 ° C / s; 85-25 ° C, cooling at 0.1 ° C / s; 4 ° C hold.
  • the present invention has the following advantages and effects:
  • the function of the antibody is only a temporary blocking effect.
  • the present invention can achieve a permanent effect;
  • the present invention can both knock out multiple coding sequences of MER6;
  • Antibodies can only target extracellular targets, and the present invention can target both extracellular and intracellular targets;

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Abstract

提供了CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA，通过计算机模拟、计算和设计，合成了一组利用CRISPR-Cas9特异性敲除人MER6基因中特异性靶向MER6基因的sgRNA，并分别将该sgRNA与线性的PX330质粒连接成载体，转染细胞即可实现MER6基因的敲除。sgRNA具有只需要少量合成多核苷酸片断，就能进行大批量生产的优点，该制备方法简单易行、sgRNA靶向性好，CRISPR-Cas9系统的敲除效率高。

Description

CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA 技术领域
本发明属于基因编辑技术领域，特别涉及一种CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA。
 背景技术
MER6是免疫球蛋白超家族成员，广泛地表达于细胞的表面，可与信号调节蛋白α、血小板反应蛋白以及整合素相互作用，介导凋亡、增殖、免疫等一系列的反应。
 技术问题
MER6是是癌细胞免于宿主免疫系统攻击的保护性受体，研究表明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，但现有技术中缺乏提升MER6基因表达的质粒，对相关研究的进展造成了一定的阻碍。
 技术解决方案
本发明的目的在于针对现有技术的缺陷和不足，提供一种结构简单，设计合理、使用方便的CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA，它通过计算机模拟、计算和设计、合成了一组利用CRISPR-Cas9特异性敲除人MER6基因中特异性靶向MER6基因的sgRNA，并分别将该sgRNA与线性的PX330质粒连接成载体，转染细胞即可实现MER6基因的敲除，可实现永久的效果；提供了高效的sgRNA；sgRNA只需要小量合成多核苷酸片断，就能大批量生产的优点。
为实现上述目的，本发明采用的技术方案是 ：
本发明所述的 CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA，它采用如下的技术方案 ：
1、sgRNA 寡核苷酸的高通量设计和选择
通过在线设计平台（http://crispr-era.stanford.edu/）设计sgRNA。
2、构建 sgRNA 的寡聚核苷酸双链
根据选择的sgRNA，在其5’端加上CCGG得到正向寡核苷酸；根据选择的sgRNA，获得其对应DNA的互补链，并且在其5’端加上AAAC得到反向寡核苷酸。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸的正反向序列寡核苷酸序列成对变性、退火，退火之后形成可以连入PX330载体的双链退火产物。
3、sgRNA 寡聚核苷酸质粒的构建
① 线性化 PX330质粒；
② 将退火的 sgRNA 寡聚核苷酸双链与线性化 PX330质粒质粒连接获得PX330-MER6质粒。
③ 转化并涂氨苄平板 (100 μg/ml)。
④ 测序鉴定阳性克隆。
⑤ 37℃摇床摇菌过夜并无内毒素抽提PX330-MER6质粒。
4、转染细胞获得 MER6基因敲除细胞
① 按照 Lipofectamine 3000 Transfection Reagent（Invitrogen）的操作手册，将带有对应sgRNA寡聚核苷酸的PX330-MER6载体转染细胞。
②用T7EN1酶切检测和TA克隆测序确认MER6基因已经被敲除。
本发明还提供了特异性靶向MER6基因的sgRNA，其序列如SEQ ID NO. 1所示。
 有益效果
本发明相对于现有技术，具有如下的优点及效果：
1、抗体的作用只是暂时封闭的作用，本发明直接敲除MER6基因，可以实现永久的效果；
2、抑制性受体有多种，如何利用多种抗体封闭多种抑制性受体还没有对策，本发明既可以针对MER6的多个编码序列进行敲除；
3、有效的MER6抗体研发困难，本发明提供了针对人MER6基因的一组高效的sgRNA；
4、抗体作用只能针对细胞外靶点，本发明既可以针对胞外，也能针对胞内靶点；
5、开发抗体药物是一项费时、费力、费钱的过程，使得抗体药物昂贵等，利用sgRNA只需要小量合成多核苷酸片断，就能大批量生产。
 附图说明
图1是PX330载体的结构；
图2是T7EN1酶切鉴定sgRNA/Cas9介导的基因人MER6特异性切割，其中，1-未经处理的对照组293T细胞，2-转染PX330-MER6载体的实验组293T细胞。
 本发明的实施方式
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。
实施例中所使用的细胞株均购自ATCC，PX330载体购自Addgene， Bbs I酶购自Thermo，Endo-Free Plasmid Mini Kit购自Omega-biotek，Lipofectamine 3000购自Invitrogen，T4 DNA连接酶购自NEB，高保真PCR酶购自Promega。
实施例一：靶向人 MER6 基因的 sgRNA 寡 聚核苷酸的合成和构建
根据选择的sgRNA序列（SEQ ID No. 1），在其5’端加上CACC得到正向寡核苷酸；根据选择的sgRNA，获得其互补链，并且在其5’端加上AAAC得到反向寡核苷酸。分别合成上述正向寡核苷酸和反向寡核苷酸，将合成的sgRNA寡聚核苷酸的正反向寡核苷酸成对变性、退火，退火之后形成可以连入PX330载体的双链sgRNA寡聚核苷酸。
变性、退火体系为 ：
2.5 μL正向寡核苷酸（100 μmol/L）
2.5 μL反向寡核苷酸（100 μmol/L）
5 μL 灭菌ddH2O
在PCR仪中按照以下程序运行：95℃，3min；95-85℃，按2℃/s降温；85-25℃，按0.1℃/s降温；4℃ hold。
实施例二：利用 CRISPR-Cas9 特异性敲除人 MER6 基因
1、线性化PX330质粒。
酶切体系和条件如下 ：
2 μg PX330-MER6；
1 μL FastDigest Buffer ；
1 μL Bbs I；
补足ddH2O至50 μL，37℃孵育1 h。
酶切完成后用 PCR Clean up Kit（天根）纯化。
2、将变性、退火之后获得的可以连入PX330载体的双链sgRNA寡聚核苷酸与线性化的 PX330质粒相连获得PX330-MER6质粒。
连接体系如下 ：
3 μL，50 μmol/L纯化退火产物
50 μg线性化的PX330质粒
1 μL 10 × T4 Ligation Buffer
1 μL T4 DNA Ligase（NEB）
ddH2O补足至10 μL
16℃孵育过夜。
4、将上述步骤获得的连接产物转化 DH5a 感受态细胞并涂氨苄平板（100 μg/mL），并挑取克隆。
5、用测序的方法鉴定获得阳性克隆。
6、37℃摇床摇菌过夜培养阳性克隆，用Endo-Free Plasmid Mini Kit无内毒素质粒抽提试剂盒抽提质粒，获得PX330-MER6质粒。
7、细胞培养与转染
① 将293T细胞接种培养于含10% FBS的DMEM培养基中。
② 在转染前分至6孔板中，待细胞融合度达到70％-80％时进行转染。
③ 按照Lipofectamine 3000 Transfection Reagent（Invitrogen）的操作手册，将2 μg的PX330-MER6质粒转染至每孔细胞中，6-8小时后换液，48小时后收取细胞。
8、T7EN1 酶切检测
① 收集转染PX330-MER6质粒的293T细胞，提取基因组DNA。
② 以提取的基因组DNA为模板，高保真PCR酶扩增后，PCR cleanup纯化后获得PCR回收产物。取100 ng统一稀释到20 μL进行变性、退火，程序为：95℃，3min；95-85℃，按2℃/s降温；85-25℃，按0.1℃/s降温；4℃ hold。
③ 在20 μL体系中加入T7EN1 0.3μL，37℃酶切30分钟后，加入2 μL 10× Loading Buffer，用1％的琼脂糖胶电泳检测，结果表明MER6基因敲除成功，如图2所示。
 工业实用性
本发明相对于现有技术，具有如下的优点及效果：
1、抗体的作用只是暂时封闭的作用，本发明直接敲除MER6基因，可以实现永久的效果；
2、抑制性受体有多种，如何利用多种抗体封闭多种抑制性受体还没有对策，本发明既可以针对MER6的多个编码序列进行敲除；
3、有效的MER6抗体研发困难，本发明提供了针对人MER6基因的一组高效的sgRNA；
4、抗体作用只能针对细胞外靶点，本发明既可以针对胞外，也能针对胞内靶点；
5、开发抗体药物是一项费时、费力、费钱的过程，使得抗体药物昂贵等，利用sgRNA只需要小量合成多核苷酸片断，就能大批量生产。

Claims (4)

  1. CRISPR-Cas9特异性敲除人MER6基因的方法以及用于特异性靶向MER6基因的sgRNA，其特征在于：
    （1）所述sgRNA在MER6基因上的靶序列符合5’-GN(20)GG-3’或者5’-N(21)GG-3’的序列排列规则；
    （2）所述sgRNA在MER6基因上的靶序列位于基因的外显子；
    （3）所述sgRNA在MER6基因上的靶序列是唯一的。
  2. 根据权利要求1所述的CRISPR-Cas9特异性敲除人MER6基因的方法以及用于特异性靶向MER6基因的sgRNA，其特征在于，其对应的DNA序列如序列表SEQ ID NO. 1所示。
  3. 根据权利要求 1 所述的 CRISPR-Cas9 特异性敲除人 MER6 基因的方法，其特征在于 ：
    （1）权利要求1-2任意一项所述的sgRNA，在其对应DNA序列的5’端加上CACC，合成得到正向寡核苷酸；权利要求1-2任意一项所述的sgRNA，获得其对应DNA序列的互补链，并且在互补链的5’端加上AAAC合成得到反向寡核苷酸；将合成的一对互补的sgRNA寡聚核苷酸的正反向寡核苷酸成对变性、退火，形成可以连入PX330基因编辑载体的双链sgRNA寡聚核苷酸；
    （2）用线性化PX330基因编辑载体；将退火的双链sgRNA寡聚核苷酸与用Bbs I线性化PX330基因编辑载体连接获得PX330-MER6载体，然后转化STBL3感受态细菌并涂氨苄平板，通过测序鉴定出阳性克隆；37℃摇床摇阳性克隆菌过夜并无内毒素抽提PX330-MER6载体；
    （3）用脂质体混合PX330-MER6载体，转染细胞；
    （4）用 T7EN1 酶切检测确认MER6基因已经被敲除并获得基因敲除的细胞。
  4. 根据权利要求3所述的 CRISPR-Cas9 特异性敲除人 MER6 基因的方法，其特征在于：步骤（3）所述的脂质体为 Lipofectamine 3000（Invitrogen）。
PCT/CN2018/091678 2018-06-15 2018-06-15 CRISPR-Cas9特异性敲除人MER6基因的方法及其特异的sgRNA WO2019237365A1 (zh)

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