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WO2019222970A1 - CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA - Google Patents

CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA Download PDF

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WO2019222970A1
WO2019222970A1 PCT/CN2018/088282 CN2018088282W WO2019222970A1 WO 2019222970 A1 WO2019222970 A1 WO 2019222970A1 CN 2018088282 W CN2018088282 W CN 2018088282W WO 2019222970 A1 WO2019222970 A1 WO 2019222970A1
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grna
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毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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    • 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

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  • the invention belongs to the field of genetic engineering and biomedical technology, and particularly relates to a CRISPR / Cas9 targeted knockout human CD226 gene and a specific gRNA thereof.
  • CD226 molecule is a widely expressed immune system such as T cells, NK cells, NK T cells, activated vascular endothelial cells, megakaryocyte / platelet lineage, monocytes, thymocytes, certain B cell subsets, some hematopoietic stem cells and mast cells.
  • T cells T cells
  • NK cells NK T cells
  • activated vascular endothelial cells megakaryocyte / platelet lineage
  • monocytes thymocytes
  • thymocytes certain B cell subsets
  • some hematopoietic stem cells and mast cells include CD226 molecule.
  • CD226 and its ligands participates in the activation, differentiation and cytotoxicity of CTL and NK cells, regulates the proliferation and differentiation of CD4 + T cells, mediates the adhesion of endothelial cells and other cells, promotes the activation and aggregation of platelets, and participates in hematopoietic regulation Mediates the degranulation of mast cells, participates in the anti-apoptotic effects of NKT cells and thymocytes, the maturation of dendritic cells, the formation of immune synapses and synapses, and has important potential in immunotherapy of tumors.
  • Intensive translational research has been conducted, but the lack of means for targeted knock-out of CD226 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a CRISPR / Cas9 targeted knockout of human CD226 gene and its specific gRNA.
  • the gRNA sequence can be used to knock out the human CD226 gene, thereby suppressing or eliminating the expression of CD226.
  • the present invention has the following advantages and effects:
  • the invention designs and synthesizes two single-stranded oligo sequences according to the gRNA guide sequence, anneals to form a double strand, and then connects with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will find a match under the guidance of the gRNA.
  • the DNA sequence was cut to achieve the CD226 gene knockout.
  • Figure 1 shows the Western Blot results of Jurkat cells in the control and experimental groups.
  • human CD226 gene was found in GenBank, and potential target sites were designed in the exon region of human CD226 gene.
  • online design tools and gRNA design principles we evaluated the high-scoring target sites on the human CD226 gene sequence to design the gRNA.
  • the sequence is shown in SEQ ID NO.1, and then the CACC was added to the 5 ′ end to obtain the positive oligo. Nucleotides, and add AAAC to the 5 'end of its reverse complement.
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule that can be ligated into the px459 vector.
  • the px459 vector was treated with Bbs I enzyme at 37 ° C for 1 h, and then electrophoresed on 1% agarose to recover the digested product.
  • the double-stranded DNA molecule that can be ligated into the px459 vector obtained in Example 1 was ligated with the px459 vector using T4 DNA ligase.
  • the ligation system (10 ⁇ l) was: annealed double stranded (CD226-gRNA) 2 ⁇ l, px459 vector 2 ⁇ l, 10 ⁇ T4 DNA Ligase Buffer 1 ⁇ l, T4 DNA Ligase 1 ⁇ l, ddH2O made up to 10 ⁇ l; connection conditions: 16 ° C overnight.
  • the ligation product is transformed into competent cells Stbl3.
  • the specific transformation method is: take out the competent cells Stbl3 at -80 ° C, and dissolve them in an ice bath; then take 1 ⁇ l of the above-mentioned ligation products to 50 ⁇ l of competent cells and mix for 30 minutes on ice; Do not shake during 42 s water bath for 60 s; cool in ice bath for 2 min; then add 800 ⁇ l LB medium and shake at 37 °C for 30 min; LB plate coated with 100 ⁇ g / ml ampicillin and culture overnight. After picking positive clones Shake at 37 ° C overnight for expansion and send for sequencing. The correct sequencing was the Cas9 vector required to target the CD226 gene, and was named px459-CD226 vector.
  • the correct strain was sequenced and identified in Example 2 and placed in an LB liquid medium having an ampicillin concentration of 100 ⁇ g / ml, and cultured at 250 rpm and 37 ° C. with shaking for 12-16 hours. Collect the bacterial solution by centrifugation at 10,000 rpm at 4 ° C, discard the supernatant, collect the bacterial cells, and then extract the plasmid according to the instructions of the Endo-Free Plasmid Mini Kit kit to obtain the px459-CD226 vector without endotoxin.
  • Jurkat cells were resuscitated. The cells were placed in a 10% FBS + DMEM culture flask and cultured in a 37 ° C, 5% CO2 incubator. One day before transfection, the recovered cells were subcultured.
  • Jurkat cells without any treatment were used as the control group, and the cells obtained in Example 4 were used as the experimental group.
  • 100-200 ⁇ l of 5 ⁇ SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes.
  • 15 ⁇ l of the loaded SDS- PAGE protein electrophoresis After electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skimmed milk powder for 2 h, place the blocked PVDF membrane in rabbit anti-human CD226 antibody, rinse the buffer 3 times, and then transfer the membrane to goat anti-rabbit secondary antibody Buffer, incubate at room temperature for 60 min, and rinse 4 times with rinsing buffer.
  • the present invention has the following advantages and effects:
  • the invention designs and synthesizes two single-stranded oligo sequences according to the gRNA guide sequence, anneals to form a double strand, and then connects with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will find a match under the guidance of the gRNA.
  • the DNA sequence was cut to achieve the CD226 gene knockout.

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Abstract

提供了一种靶向敲除人CD226基因的特异性gRNA，根据CRISPR/Cas9 gRNA的设计原则，在人CD226基因上设计了两个靶位点，合成相应的寡聚核苷酸，并且将其构建在px459载体上。在Jurkat细胞中利用该重组载体指导的CRISPR/Cas9系统，可以有效地敲除人CD226基因。提供的gRNA指导CRISPR/Cas9系统有望在治疗肿瘤的新型药物中得到应用。

Description

CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA 技术领域
本发明属于基因工程与生物医学技术领域，特别涉及一种CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA。
 背景技术
CD226分子是一种广泛表达T细胞、NK细胞、NK T细胞、活化的血管内皮细胞、巨核/血小板谱系、单核细胞、胸腺细胞、某些B细胞亚群、部分造血干细胞和肥大细胞等免疫细胞表面的Ⅰ型跨膜糖蛋白。
 技术问题
CD226与其配体的相互作用参与CTL和NK细胞的活化、分化和细胞毒作用，调节CD4+ T细胞的增殖和分化，介导内皮细胞与其他细胞的黏附，促进血小板的活化与聚集，参与造血调控，介导肥大细胞的脱颗粒作用，参与NKT细胞和胸腺细胞的抗凋亡作用、树突状细胞的成熟、免疫突触和神经突触的形成，在肿瘤的免疫治疗中具有重要潜力，需进行深入的转化研究，但现有技术中缺乏靶向敲除CD226基因表达的手段，对相关研究的进展造成了一定的阻碍。
 技术解决方案
针对上述问题，本发明提供一种CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA，该gRNA序列可以用于敲除人CD226基因，进而抑制或消除CD226的表达。
本发明申请的技术方案如下：
1、靶向人CD226基因的高效gRNA设计合成以及gRNA/cas9表达系统构建。
2、在Jurkat细胞中分析检测本发明gRNA指导的CRISPR/Cas9系统对于CD226基因的抑制作用。
 有益效果
本发明相对于现有技术，具有如下的优点及效果：
本发明根据gRNA导向序列设计合成两条单链oligo序列，退火形成双链，然后与Cas9载体连接，利用Cas9载体将gRNA以及CRISPR系统引入目标细胞中，Cas9蛋白会在gRNA的引导下找到与其匹配的DNA序列，进行剪切，实现CD226基因的敲除。
 附图说明
图1为对照组和实验组Jurkat细胞的Western Blot结果图。
 本发明的实施方式
实施例中所使用的细胞株均购自ATCC，px459载体购自Addgene，内切酶Bbs I购自Thermo，Endo-Free Plasmid Mini Kit购自Omega-biotek，Lipofectamine 2000购自Invitrogen，T4 DNA连接酶购自NEB，嘌呤霉素购自Sigma。
实施例一 靶向 CD226 基因的 gRNA 设计
在GenBank中找到人CD226基因的序列，在人CD226基因的外显子区域设计潜在靶位点。通过在线设计工具及gRNA的设计原则，评估人CD226基因序列上得分较高的靶位点设计gRNA，其序列如SEQ ID NO.1所示，然后在其5 '端加上CACC得到正向寡核苷酸，并且在其反向互补序列的5 '端加上AAAC。分别合成上述正向寡核苷酸和反向寡核苷酸，95℃变性，退火，形成可连入px459载体的双链DNA分子。
实施例二 构建表达 gRNA 的载体
用Bbs I酶，37℃处理px459载体1 h后，1％的琼脂糖电泳，回收酶切产物。用T4 DNA 连接酶将实施例一中获得的可连入px459载体的双链DNA分子与px459载体进行连接。连接体系（10 μl）为：退火双链(CD226-gRNA) 2 μl，px459载体2 μl，10 × T4 DNA Ligase Buffer 1 μl，T4 DNA Ligase 1 μl，ddH2O补足至10 μl；连接条件：16℃连接过夜。
将连接产物转化感受态细胞Stbl3，具体转化方法为：-80℃取出感受态细胞Stbl3，冰浴溶解；然后取50 μl感受态细胞中加入1 μl的上述连接产物，混匀后冰浴30min；42℃水浴60 s，过程中勿摇动；冰浴冷却2 min；然后加入800 μl LB培养基，37℃摇床30min；涂含100 μg/ml氨苄青霉素的LB板培养过夜，挑取阳性克隆后37℃摇床过夜进行扩大培养并送测序。测序正确的即为所需的靶向CD226基因的Cas9载体，命名为px459-CD226载体。
实施例三 无内毒素质粒 DNA 的制备
取实施例二中测序鉴定正确的菌株，置于氨苄青霉素浓度为100 μg/ml的LB液体培养基中，250 rpm、37℃振荡培养12-16 h。4℃，10000 rpm离心收集菌液，弃上清，收集菌体，然后按照Endo-Free Plasmid Mini Kit试剂盒说明书操作步骤提取质粒，得无内毒素的px459-CD226载体。
实施例四Jurkat 细胞的转染
转染前3天，复苏Jurkat细胞，将细胞放入加有10％的FBS+DMEM培养瓶中，于37℃、5％CO2的培养箱中培养，转染前一天，传代培养复苏细胞。
将培养Jurkat细胞T75瓶中的培养基吸净，加入2 ml 4℃冰箱取出的0.25％胰酶，使其均匀覆盖瓶底，置于37℃培养箱中3-5 min，取出，摇晃可发现细胞于底部脱离，将其全部晃下，加入3 ml 37℃水浴中预热的10％DMEM，用10 ml移液管进行吹打，吹打6-8次，不留死角，瓶口处较难吹打可将移液管对准培口，小力将培养基打出即可覆盖到接近瓶口的细胞。之后，将所有细胞吸出，置于15 ml离心管中，取50ul混匀后的细胞于1.5 ml EP管中，加入450 μl 10％ DMEM，即为10倍稀释，混匀，取10 μl细胞于计数板中计数。传代当天记为第一天，若第二天进行转染，铺9-10×10 7/T75；若第三天转染，铺3.5-4×10 7/T75。每瓶T75加15 ml 10％DMEM培养基。转染当天观察细胞密度，融合度达到80-90％满可进行转染。按Lipofectamine 2000说明书的步骤，将px459-CD226转染Jurkat细胞。
转染48小时后，利用胰酶消化转染后贴壁的细胞，离心收集细胞，吸掉废液加入1 ml PBS重悬细胞，取500 μl放入原瓶中继续培养，剩余细胞放入1 .5ml离心管，提取总蛋白。
实施例五 Western Blot 检测转染效果
以未经任何处理的Jurkat细胞作为对照组，实施例四中获得的细胞为实验组，分别加入100-200μl 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CD226抗体，缓冲液漂洗3次后，再将膜转移至山羊抗兔二抗缓冲液，室温孵育60 min，再用漂洗缓冲漂洗4次。漂洗完毕后将蛋白印迹膜用ECL显影检测，结果如图1所示。可以看到，CD226移码基因突变Jurkat细胞中Western Blot检测不到CD226蛋白条带，而对照组则有CD226蛋白条带出现，说明所述用于敲除人细胞CD226基因的gRNA序列可以实现CD226基因的敲除。
 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：
本发明根据gRNA导向序列设计合成两条单链oligo序列，退火形成双链，然后与Cas9载体连接，利用Cas9载体将gRNA以及CRISPR系统引入目标细胞中，Cas9蛋白会在gRNA的引导下找到与其匹配的DNA序列，进行剪切，实现CD226基因的敲除。

Claims (3)

  1. 在CRISPR/Cas9特异性敲除人CD226基因中用于特异性靶向人CD226基因的gRNA，其特征在于，所述gRNA在人CD226基因上的靶序列符合5’-N(20)-NGG3’或者5’-CCN-N(20)-3’的序列排列规则，在人CD226基因上的靶序列是唯一的。
  2. 根据权利要求1所述的在CRISPR/Cas9特异性敲除人CD226基因中用于特异性靶向人CD226基因的gRNA，其特征在于，所述靶向位点如SEQ ID NO .1所示。
  3. 根据权利要求2所述的在CRISPR/Cas9特异性敲除人CD226基因中用于特异性靶向人CD226基因的gRNA，其特征在于：所述的肿瘤细胞为Jurkat细胞。
PCT/CN2018/088282 2018-05-24 2018-05-24 CRISPR/Cas9靶向敲除人CD226基因及其特异性gRNA Ceased WO2019222970A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651392A (zh) * 2015-01-06 2015-05-27 华南农业大学 一种利用CRISPR/Cas9系统定点突变P/TMS12-1获得温敏不育系的方法
CN105637087A (zh) * 2013-09-18 2016-06-01 科马布有限公司 方法、细胞与生物体
CN105647969A (zh) * 2016-02-16 2016-06-08 湖南师范大学 一种基因敲除选育stat1a基因缺失型斑马鱼的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105637087A (zh) * 2013-09-18 2016-06-01 科马布有限公司 方法、细胞与生物体
CN104651392A (zh) * 2015-01-06 2015-05-27 华南农业大学 一种利用CRISPR/Cas9系统定点突变P/TMS12-1获得温敏不育系的方法
CN105647969A (zh) * 2016-02-16 2016-06-08 湖南师范大学 一种基因敲除选育stat1a基因缺失型斑马鱼的方法

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