WO2019237385A1 - 用于敲除人TNFSF9基因的gRNA序列及其应用 - Google Patents

用于敲除人TNFSF9基因的gRNA序列及其应用 Download PDF

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WO2019237385A1
WO2019237385A1 PCT/CN2018/091715 CN2018091715W WO2019237385A1 WO 2019237385 A1 WO2019237385 A1 WO 2019237385A1 CN 2018091715 W CN2018091715 W CN 2018091715W WO 2019237385 A1 WO2019237385 A1 WO 2019237385A1
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tnfsf9
gene
knocking out
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tnfsf9 gene
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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
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

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  • the invention belongs to the technical field of genetic engineering and gene editing, and particularly relates to a gRNA sequence for knocking out the human TNFSF9 gene and application thereof.
  • CDw137 belongs to the TNFR superfamily and is mainly expressed in activated T cells and is an inducible T cell surface receptor.
  • TNFSF9 belongs to the TNF superfamily and is mainly expressed in concentrated antigen presenting cells (APC).
  • APC concentrated antigen presenting cells
  • CDw137 / TNFSF9 is another important costimulatory molecule besides CD28 / B7, which can or does not depend on the CD28 / B7 pathway to mediate the production of co-stimulatory signals, induce T cell activation, proliferation and cytokine secretion.
  • CDw137 and its ligand system have two-way signal transduction, which can not only transmit cells to T cells through TNFSF9, but also transmit signals to cells expressing ligands. It plays an important role in the immunotherapy of tumors and requires a lot of research methods. Clinical transformation can be achieved, but the lack of means for knocking out TNFSF9 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • Regularly spaced clustered short palindrome repeats Regularly Interspaced Short Palindromic Repeats is a series of clustered DNA Sequences are a system that bacteria use to protect themselves against viruses and a genetic weapon against attackers.
  • the Cas gene encodes a protein that contains nucleases, polymerases, helicases, and domains that bind to ribonucleic acid.
  • RNA transcribed by CRISPR combines with the Cas protein to form a ribonucleoprotein complex that cooperates with the immune function of the CRISPR / Cas system to guide the Cas protein. Therefore, this RNA is also called guide RNA.
  • the Cas protein in the complex can cut the invading virus DNA to achieve the purpose of defense. Therefore, you only need to synthesize a guide RNA-oriented DNA sequence for the DNA sequence that needs to be edited.
  • the artificially constructed gRNA can guide the Cas9 protein to accurately cut the specific DNA sequence of the host cell to play a gene. The role of the editor.
  • the purpose of the present invention is to overcome the defects existing in the prior art, provide a gRNA sequence that knocks out the human TNFSF9 gene, and construct a corresponding gene knockout vector px458-TNFSF9, which lays a foundation for the subsequent research on the function of the human TNFSF9 gene.
  • a gRNA sequence that knocks out the TNFSF9 gene in human cells and uses thereof include the following steps:
  • the transfected cell culture medium was changed to a serum-free medium, and the px458-TNFSF9 recombinant plasmid was prepared into a transfection mixture with Opti-MEM medium and PEI, added to the transfected cell culture medium, and changed after 5 h. After transfecting the cell culture medium with serum medium and continuing the culture for 48 h, the transfected cells with TNFSF9 gene knocked out were obtained.
  • the gRNA sequence of the human TNFSF9 gene knockout provided by the present invention and its application can play an important role in the research and development of TNFSF9-related drugs.
  • Figure 1 is a plasmid map of the px458 vector
  • FIG. 2 shows the results of Western Blot detection of TNFSF9 protein in control and experimental K562 cells.
  • E. coli NEBStable and T4 DNA ligase were purchased from NEB, px458 plasmid was purchased from Addgene, Bbs I endonuclease was purchased from Fermentas, PEI was purchased from Sichuan Best, Opti-MEM medium was purchased from Invitrogen, endotoxin-free plasmid extraction reagent The Endo-Free Plasmid Mini Kit was purchased from Omega bio-tek.
  • Each of the two nucleotide sequences was prepared to 100 ⁇ mol / l with deionized bacteria water, placed in 600 ml of boiling water, and cooled and annealed at room temperature to form a double-stranded gRNA sequence.
  • a 100 ⁇ g / ml ampicillin-containing LB medium was used to incubate the correctly sequenced E. coli at 37 ° C.
  • the px458-TNFSF9 recombinant plasmid was extracted without endotoxin.
  • K562 cells were seeded into a six-well plate one day before transfection at a seeding density of 50%.
  • Transduction Take 1 ⁇ g px458-TNFSF9 recombinant plasmid and 3 ⁇ l PEI (1 ⁇ g / ⁇ l) dissolved in 100 ⁇ l Opti-MEM medium, vortex and mix. The medium in the six-well plate was changed to serum-free medium, 2 ml per well, 600 ⁇ l per well was added to the transfection mixture, and 5 hours was replaced with DMEM medium preheated with 10% fetal bovine serum at 37 ° C. Incubate at 37 ° C for 5% CO2, 90% humidity for 48 h, and collect cells.
  • the genomic DNA of the cells collected in Example 2 was extracted, and then sequencing primers were designed to sequence the genomic DNA at the target location based on the sequence of the human TNFSF9 DNA 205-352 bp position.
  • K562 cells without any treatment were used as the control group, and the K562 cells collected in Example 2 were used as the experimental group.
  • 100-200 ⁇ l of 5 ⁇ SDS-PAGE loading buffer was added and boiled in boiling water for 5 minutes. Min, take 15 ⁇ l of loaded SDS-PAGE protein for electrophoresis. After the electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human TNFSF9 antibody (1: 1000, Abcam / ab68185), and the buffer solution was 10% skim milk powder.
  • TNFSF9 protein band cannot be detected by Western Blot in the TNFSF9 frameshift mutant K562 cells, while the control group has a TNFSF9 protein band, indicating that the gRNA sequence used to knock out the TNFSF9 gene of human cells can achieve TNFSF9. Gene knockout.
  • the gRNA sequence of the human TNFSF9 gene knockout provided by the present invention and its application can play an important role in the research and development of TNFSF9-related drugs.

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Abstract

提供了一种用于敲除人细胞TNFSF9基因的gRNA序列。在细胞中该gRNA能够特异结合于人TNFSF9DNA正义链第261-280bp，并与Cas9形成复合物，对TNFSF9基因核酸序列进行特异切割，利用细胞非重组末端连接修复机制，造成TNFSF9基因移码突变，进而获得TNFSF9基因敲除细胞。

Description

用于敲除人TNFSF9基因的gRNA序列及其应用 技术领域
本发明属于基因工程和基因编辑技术领域，尤其涉及一种用于敲除人TNFSF9基因的gRNA序列及其应用。
 背景技术
CDw137属于TNFR超家族，主要表达于活化的T细胞中，是一种可诱导的T细胞表面受体；TNFSF9属于TNF超家族，主要表达在集中抗原呈递细胞（APC）。CDw137/TNFSF9是CD28/B7之外的另一重要的共刺激分子，可依赖或不依赖于CD28/B7途径而介导产生协同刺激信号，诱导T细胞的活化、增殖与细胞因子的分泌。
 技术问题
CDw137及其配体系统存在双向信号传导，既可通过TNFSF9向T细胞传递细胞，又可将信号传向表达配体的细胞，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，但现有技术中缺乏敲除TNFSF9基因表达的手段，对相关研究的进展造成了一定的阻碍。
规律间隔成簇短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats，CRISPR）是一系列成簇排列的DNA 序列，是细菌用以保护自身对抗病毒的一个系统，也是一种对付攻击者的基因武器。Cas基因编码的蛋白包含核酸酶、聚合酶、解旋酶以及与核糖核酸结合的结构域。
CRISPR转录出的RNA与Cas蛋白结合形成核糖核蛋白复合物协同行使CRISPR/Cas系统的免疫功能，来对Cas蛋白起到导向作用，因此这段RNA也被称为导向RNA（guide RNA）。当入侵的病毒DNA 和gRNA 序列一致时，复合物中的Cas蛋白就能够切割入侵的病毒DNA，达到防御的目的。因此，只需针对需要编辑的DNA 序列合成一段导向RNA的DNA序列，在转入宿主细胞后，产生的人工构建的gRNA就能指导Cas9蛋白精准地切割宿主细胞特定的DNA 序列，从而起到基因编辑的作用。
 技术解决方案
本发明的目的在于克服现有技术中的存在的缺陷，提供一种敲除人TNFSF9基因的gRNA序列，并构建相应基因敲除载体px458-TNFSF9，为后续研究人TNFSF9基因功能奠定基础。
其具体技术方案为：
一种敲除人细胞TNFSF9基因的gRNA序列及其应用，包括以下步骤：
（1）人工合成所述靶DNA序列及其互补链，退火后将核酸片段重组至px458质粒中获得px458-TNFSF9重组质粒，转化大肠杆菌NEBStable，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定；
（2）大量培养测序正确的大肠杆菌，无内毒素提取px458-TNFSF9重组质粒；
（3）将转染细胞培养基换为无血清培养基，将px458-TNFSF9重组质粒用Opti-MEM培养基和PEI配制成转染混合液，加入到转染细胞培养基中，5 h后换液；转染细胞培养基替换为血清培养基继续培养48 h后，即得敲除TNFSF9基因的转染细胞。
 有益效果
本发明提供的敲除人TNFSF9基因的gRNA序列及其应用，可在TNFSF9相关的药物研究和开发中将起重要作用。
 附图说明
图1 为px458载体的质粒图谱；
图2 为对照组和实验组K562细胞的TNFSF9蛋白的Western Blot检测结果图。
 本发明的实施方式
下面结合附图与具体实施例对本发明做进一步的说明。
大肠杆菌NEBStable和T4 DNA连接酶购自NEB，px458质粒购自Addgene，Bbs I内切酶购自Fermentas，PEI购自四川贝思特，Opti-MEM培养基购自Invitrogen，无内毒素质粒提取试剂盒Endo-Free Plasmid Mini Kit 购自Omega bio-tek公司。
实施例一 px458-TNFSF9 质粒构建
合成核苷酸序列5’- CACCGGGATCGTCGGGCGAAAGCT -3’，及其反向互补序列5’- AAACGGGATCGTCGGGCGAAAGCT -3’。将两种核苷酸序列各自用去离子菌水配成100 μmol/l，置600 ml 沸水，自然室温冷却退火，形成双链gRNA序列。
Bbs I双酶切px458 质粒，回收后将其与所述gRNA序列按1:5混合后，T4 DNA连接酶16℃连接过夜。转化大肠杆菌NEBStable，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定。
实施例二 K562 细胞转导
用含100 μg/ml氨苄青霉素的LB培养基，37℃大量培养测序正确的大肠杆菌，无内毒素提取px458-TNFSF9重组质粒。
转染前一天将K562细胞接种至六孔板，接种密度50%。转导：取1 μg px458-TNFSF9重组质粒和3 μl PEI（1 μg/μl）溶于100 μl Opti-MEM培养基，涡旋混匀。将六孔板内培养基换成无血清培养基，每孔2 ml，每孔600 μl加入转染混合液，5 h换成37℃预热有10% 胎牛血清的DMEM培养基。置37℃ 5% CO2，90% 湿度培养48 h，收集细胞。
实施例三   细胞 TNFSF9 突变鉴定
提取实施例二中收集细胞的基因组DNA，然后根据人TNFSF9 DNA 205-352 bp位置的序列设计测序引物对基因组DNA进行测序，在靶点位置。
实施例四   细胞 TNFSF9 蛋白表达的检测
以未经任何处理的K562细胞作为对照组，实施例二中收集的K562细胞为实验组，分别加入100-200 μl 5 × SDS-PAGE上样缓冲液，沸水煮5 min，取15 μl 上样SDS-PAGE 蛋白电泳。电泳完毕后，按照常规蛋白半干转，10%脱脂奶粉封闭2 h，将封闭后的PVDF膜置于兔抗人TNFSF9 抗体（1:1000，Abcam/ab68185），缓冲液为10%脱脂奶粉，冰上孵育过夜，然后用漂洗缓冲液漂洗四次，每次5 分钟，再将膜转移至0.05M PBS、PH 7.2、0.2% Tween-20、1:50000稀释山羊抗兔二抗缓冲液（Abcam/ ab97051），室温孵育60 min，再用漂洗缓冲漂洗四次，每次5min。漂洗完毕后将蛋白印迹膜用ECL显影检测，结果如图2所示。可以看到，TNFSF9移码基因突变K562细胞中Western Blot检测不到TNFSF9蛋白条带，而对照组则有TNFSF9蛋白条带出现，说明所述用于敲除人细胞TNFSF9基因的gRNA序列可以实现TNFSF9基因的敲除。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
 工业实用性
本发明提供的敲除人TNFSF9基因的gRNA序列及其应用，可在TNFSF9相关的药物研究和开发中将起重要作用。

Claims (3)

  1. 一种用于敲除人细胞TNFSF9基因的CRISPR/Cas9 guide RNA的靶DNA序列，所述序列为5’- GGGATCGTCGGGCGAAAGCT -3’。
  2. 一种采用权利要求1 所述DNA 序列敲除人细胞TNFSF9基因的方法，具体步骤如下：
    （1）人工合成所述靶DNA序列及其互补链，退火后将核酸片段重组至px458质粒中获得px458-TNFSF9重组质粒，转化大肠杆菌NEBStable，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定；
    （2）大量培养测序正确的大肠杆菌，无内毒素提取px458-TNFSF9重组质粒；
    （3）将转染细胞培养基换为无血清培养基，将px458-TNFSF9重组质粒用Opti-MEM培养基和PEI配制成转染混合液，加入到转染细胞培养基中，5 h后换液；转染细胞培养基替换为血清培养基继续培养48 h后，即得敲除TNFSF9基因的转染细胞。
  3. 权利要求1 所述DNA 序列在敲除TNFSF9基因中的应用。
PCT/CN2018/091715 2018-06-16 2018-06-16 用于敲除人TNFSF9基因的gRNA序列及其应用 WO2019237385A1 (zh)

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