WO2019237398A1 - 一种特异靶向人CD272基因的gRNA导向序列及其应用 - Google Patents

一种特异靶向人CD272基因的gRNA导向序列及其应用 Download PDF

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WO2019237398A1
WO2019237398A1 PCT/CN2018/091728 CN2018091728W WO2019237398A1 WO 2019237398 A1 WO2019237398 A1 WO 2019237398A1 CN 2018091728 W CN2018091728 W CN 2018091728W WO 2019237398 A1 WO2019237398 A1 WO 2019237398A1
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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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  • the invention belongs to the technical field of gene editing, and particularly relates to a gRNA guide sequence specifically targeting the human CD272 gene and application thereof.
  • CD272 is the third inhibitory molecule found in the CD28 family. It is mainly expressed on the surface of B cells, T cells, and macrophages. It has a similar structure to CTLA-4 and PD-1, but their expression characteristics are different. CTLA-4 and PD-1 are not expressed on resting T cells, and the expression gradually increases after activation, while CD272 is constitutively expressed on resting T cells, and continues to be expressed after activation. CD272 signal can inhibit the proliferation of CD3 activated T cells and the secretion of IL-2 and IFN- ⁇ .
  • CD272 plays an important role in maintaining peripheral immune tolerance and transplantation immunity, and has good clinical transformation prospects, and further research is needed.
  • the existing technology lacks the means to target CD272 gene knockout. Progress has created some obstacles.
  • the present invention provides a gRNA targeting sequence that specifically targets the human CD272 gene.
  • the gRNA targeting sequence can be used to knock out the human CD272 gene, thereby suppressing or eliminating the expression of CD272.
  • Yet another object of the present invention is to provide the application of the gRNA targeting sequence specifically targeting the human CD272 gene.
  • CD272-gRNA A gRNA-directed sequence that specifically targets the human CD272 gene is CD272-gRNA, and its nucleotide sequence is:
  • CD272-gRNA 5'- TGTTCCAGATGTCCAGATAT -3 ';
  • a method for knocking out the human CD272 gene using the CRISPR / Cas9 system includes the following steps:
  • step b ligating the double strand prepared in step a with the Cas9 vector to obtain a recombinant knockout expression vector
  • the recombinant knockout expression vector prepared in step b is transfected into the target cells, and puromycin is selected to obtain cells that successfully knocked out the CD272 gene.
  • the Cas9 vector in step b is a px459 vector
  • the target cell in step c is an A549 cell
  • the concentration of puromycin in step c is 1.0 ⁇ g / ml.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell, and the Cas9 protein will be found under the guidance of the gRNA.
  • the matching DNA sequence was cut to achieve the CD272 gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.
  • Figure 1 shows the Western Blot results of A549 cells in the control and experimental groups.
  • a gRNA targeting human CD272 gene was designed.
  • the 20nt oligonucleotide gRNA targeting sequence is: CD272-gRNA: 5'- TGTTCCAGATGTCCAGATAT -3 ', then add CACC to its 5' end to obtain a forward oligonucleotide, and 5 'to its reverse complementary sequence Add AAAC to the end.
  • 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 has a Bbs I digestion site and is digested with Bbs I.
  • the digestion system (20 ⁇ l) is: Bbs I 1 ⁇ L; 10 ⁇ FastDigest Buffer 2 ⁇ L; plasmid 1 ⁇ g; ddH2O make up to 20 ⁇ l; digestion conditions: digestion at 37 ° C for 1 h. After the digestion is completed, the gel is recovered and purified.
  • the digested vector px459 and the annealed double strand obtained in Example 1 were ligated with T4 ligase.
  • the ligation system (10 ⁇ l) was: annealed double strand (CD272-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 is the required Cas9 vector to target the CD272 gene, which is named px459-CD272 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 endotoxin-free px459-CD272 vector.
  • A549 cells Cultivate A549 cells until the fusion rate of A549 cells reaches 50% to 60%.
  • the optimal transfection time is 12 to 18 hours after inoculation. Change the fresh culture medium before transfection. Add 3 ml medium to a 60 mm culture dish. Transfection At the time, 4 ⁇ g of px459-CD272 plasmid was introduced according to the instructions of the Lipofectamine 2000 kit. 48 hours after transfection, 1 ⁇ g / ml puromycin was added for screening for 7 days. After the screening was completed, the concentration of puromycin was reduced to 0.5 ⁇ g / ml and the cells were expanded.
  • A549 cells without any treatment were used as the control group, and the cells selected 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 SDS was loaded.
  • -PAGE protein electrophoresis After electrophoresis, semi-dry transfer with conventional proteins, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human CD272 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 Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 1. It can be seen that the CD272 protein band cannot be detected by Western Blot in the CD272 frameshift gene mutant A549 cells, while the CD272 protein band appears in the control group, indicating that the g272 sequence used to knock out the CD272 gene of human cells can achieve CD272. Gene knockout.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will be found under the guidance of the gRNA.
  • the matching DNA sequence was cut to achieve the CD272 gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.

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Abstract

一种特异靶向人CD272基因的gRNA导向序列及其应用，属于基因编辑技术领域。所述的gRNA导向序列的核苷酸序列为5'-TGTTCCAGATGTCCAGATAT -3'。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CD272基因的敲除。

Description

一种特异靶向人CD272基因的gRNA导向序列及其应用 技术领域
本发明属于基因编辑技术领域，特别涉及一种特异靶向人CD272基因的gRNA导向序列及其应用。
 背景技术
CD272是CD28家族发现的第3个抑制性分子，主要表达在B细胞、T细胞、巨噬细胞等细胞表面，具有和CTLA-4、PD-1相似的结构，但它们的表达特点有所不同，CTLA-4和PD-1在静止的T细胞上不表达，活化后表达逐步升高，而CD272在静止T细胞上组成性表达，活化后继续表达。CD272信号能够抑制CD3活化的T细胞增殖和IL-2、IFN-γ的分泌。
 技术问题
研究表明，CD272在维持外周免疫耐受和移植免疫中具有重要作用，具有很好的临床转化前景，需进一步研究，但现有技术中缺乏靶向敲除CD272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，本发明提供一种特异靶向人CD272基因的gRNA导向序列，该gRNA导向序列可以用于敲除人CD272基因，进而抑制或消除CD272的表达。
本发明的再一目的在于提供上述特异靶向人CD272基因的gRNA导向序列的应用。
本发明的目的通过下述技术方案实现：
一种特异靶向人CD272基因的gRNA导向序列，为CD272-gRNA，其核苷酸序列为：
CD272-gRNA：5’- TGTTCCAGATGTCCAGATAT -3’；
一种利用CRISPR/Cas9系统敲除人CD272基因的方法，包含如下步骤：
a. 在上述的gRNA导向序列的5 '端加上CACC得到正向寡核苷酸；同时根据导向序列获得其对应的DNA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，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；
b. 将步骤a制得的双链与Cas9载体连接，得到重组敲除表达载体；
c. 将步骤b制得的重组敲除表达载体转染至目的细胞中，嘌呤霉素筛选，得到成功敲除CD272基因的细胞。
进一步的，步骤b中所述Cas9载体为px459载体；
进一步的，步骤c中所述目的细胞为A549细胞；
进一步的，步骤c中所述嘌呤霉素的浓度为1.0 μg/ml。
 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、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CD272基因的敲除。
2、载体中含有嘌呤霉素抗性基因，利用嘌呤霉素对细胞进行筛选，可将未转入载体的细胞筛选淘汰。
 附图说明
图1为对照组和实验组A549细胞的Western Blot结果图。
 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，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 CD272 基因的 gRNA 设计
根据人CD272基因的基因组序列，设计1个靶向人CD272基因的gRNA。20nt的寡核苷酸gRNA导向序列为：CD272-gRNA：5’- TGTTCCAGATGTCCAGATAT -3’，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，形成可以连入px459载体的双链DNA分子。
实施例二构建表达 gRNA 的载体
px459载体有Bbs I酶切位点，用Bbs I酶切，其中酶切体系（20μl）为：Bbs Ⅰ 1μL；10× FastDigest buffer 2 μL；质粒1 μg；ddH2O补足至20 μl ；酶切条件为：37℃酶切1 h,。酶切完成后进行胶回收纯化。
将酶切后的载体px459分别与实施例一中获得的退火双链利用T4连接酶进行连接，连接体系（10 μl）为：退火双链(CD272-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CD272基因的Cas9载体，命名为px459-CD272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-CD272载体。
实施例四 A549 细胞的转染
培养A549细胞，待A549细胞的融合率达到50％～60％，接种后12～18h为最佳转染时间；转染前更换新鲜培养液，60 mm培养皿中加入3 ml培养基；转染时按照Lipofectamine 2000试剂盒说明书导入4μg的px459-CD272质粒，转染后48 h，加入1 μg/ml 嘌呤霉素筛选7 d。筛选完成后，将嘌呤霉素的浓度降为0.5 μg/ml继续扩大培养细胞。
实施例五 Western Blot 检测转染效果
以未经任何处理的A549细胞作为对照组，实施例四中筛选出的细胞为实验组，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CD272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，CD272移码基因突变A549细胞中Western Blot检测不到CD272蛋白条带，而对照组则有CD272蛋白条带出现，说明所述用于敲除人细胞CD272基因的gRNA序列可以实现CD272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：
1、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CD272基因的敲除。
2、载体中含有嘌呤霉素抗性基因，利用嘌呤霉素对细胞进行筛选，可将未转入载体的细胞筛选淘汰。

Claims (5)

  1. 一种特异靶向人CD272基因的gRNA导向序列，其特征在于：所述的特异靶向人CD272基因的gRNA导向序列为CD272-gRNA，其核苷酸序列为：
    CD272-gRNA：5’- TGTTCCAGATGTCCAGATAT -3’。
  2. 一种利用CRISPR/Cas9系统敲除人CD272基因的方法，其特征在于包含如下步骤：
    a. 在权利要求1所述的gRNA导向序列的5 '端加上CACC得到正向寡核苷酸；同时根据导向序列获得其对应的DNA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，将合成的正向寡核苷酸和反向寡核苷酸变性，退火，形成双链；
    b. 将步骤a制得的双链与Cas9载体连接，得到重组敲除表达载体；
    c. 将步骤b制得的重组敲除表达载体转染至目的细胞中，嘌呤霉素筛选，得到成功敲除CD272基因的细胞。
  3. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人CD272基因的方法，其特征在于：步骤b中所述Cas9载体为px459载体。
  4. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人CD272基因的方法，其特征在于：步骤c中所述目的细胞为A549细胞。
  5. 根据权利要求2所述的利用CRISPR/Cas9系统敲除人CD272基因的方法，其特征在于：步骤c中所述嘌呤霉素的浓度为1.0 μg/ml。
PCT/CN2018/091728 2018-06-16 2018-06-16 一种特异靶向人CD272基因的gRNA导向序列及其应用 WO2019237398A1 (zh)

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