WO2019237392A1 - CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA - Google Patents

CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA Download PDF

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WO2019237392A1
WO2019237392A1 PCT/CN2018/091722 CN2018091722W WO2019237392A1 WO 2019237392 A1 WO2019237392 A1 WO 2019237392A1 CN 2018091722 W CN2018091722 W CN 2018091722W WO 2019237392 A1 WO2019237392 A1 WO 2019237392A1
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sleb2
grna
gene
human
crispr
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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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    • C12N15/09—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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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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 genetic engineering and gene editing, and particularly relates to a CRISPR / Cas9 targeted knockout human SLEB2 gene and a specific gRNA thereof.
  • SLEB2 is an important immunosuppressive molecule and is a member of the CD28 superfamily.
  • the signaling pathway composed of SLEB2 and its ligand PDCD1L1 is one of the most important signaling pathways in the body's immune response. Its activation can lead to the formation of an immunosuppressive tumor microenvironment, allowing tumor cells to escape the body's immune surveillance and killing, and block SLEB2 / PDCD1L1 signaling pathway can reverse tumor immune microenvironment and enhance endogenous anti-tumor immune effects. Therefore, the research on the role of SLEB2 in tumor treatment and its mechanism is very important, and further research is needed.
  • Immunomodulation with SLEB2 as a target has important significance in the fight against tumors, anti-infection, anti-autoimmune diseases, and organ transplant survival. Its potential clinical transformation value is great, and solid research is required before it can be put into practical application. However, the lack of a means of knocking out SLEB2 gene expression in the prior art has caused a certain obstacle to the progress of related research.
  • the CRISPR / Cas9 gene editing system has been successfully applied to animals and plants, bacteria, yeast and other cells. It is the most advanced genome editing system currently available. It can quickly, easily, efficiently, and specifically target knockout genes for efficient targeted knockout of SLEB2. Genes that provide a possible option for the treatment of SLEB2 gene-related diseases.
  • the purpose of the present invention is to verify the efficient and targeted deletion of the SLEB2 gene by using CRISPR / Cas9, and provide corresponding technical solutions to achieve the purpose of specifically deleting the SLEB2 gene.
  • the purpose of the present invention is to provide a highly efficient gRNA and its target site sequence based on the CRISPR / Cas9 system, which simultaneously target the human SLEB2 gene, and inhibit the expression of the human SLEB2 gene through design, construction, and screening.
  • the CRISPR / Cas9 targeted knockout human SLEB2 gene and its specific gRNA provided by the present invention provide an experimental technology platform for in-depth exploration of the role of the SLEB2 gene, and can be used in research and development of drugs related to abnormal expression of the SLEB2 gene.
  • FIG 1 shows the results of the T7 endonuclease test.
  • E. coli Stbl3 was purchased from Beijing Quanshijin, T4 DNA ligase was purchased from Thermo, px459 plasmid was purchased from Addgene, Bbs I endonuclease was purchased from Fermentas, Lipofectamine 3000 and Opti-MEM medium were purchased from Invitrogen, Endo-Free Plasmid Mini Kit was purchased from Omega bio-tek company, genomic DNA extraction kit was purchased from Beijing Tiangen, T7 endonuclease I was purchased From NEB, PrimeSTAR HS (premix) was purchased from Dalian Baobi.
  • the synthesized nucleotide sequences were each made into 100 with deionized bacteria water. ⁇ mol / l, placed in 500 ml of boiling water, and cooled and annealed at room temperature to form a double-stranded gRNA sequence.
  • 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 4 ° C and 10,000 rpm, 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-SLEB2 plasmid without endotoxin.
  • Jurkat cells were seeded into a six-well plate with 500,000 cells per well and cultured until the next day. 2.5 ⁇ g of Lipofectamine 3000 was used.
  • the px459-SLEB2 recombinant vector was transduced to the Jurkat cells. After 24 hours of incubation, the transduced cells were digested into a 60 mm petri dish, and a final concentration of 1.0 ⁇ g / ml puromycin was added to the petri dish for culture, which was set as the experimental group; Transduced Jurkat was used as a control group, and cultured in a medium containing puromycin at a final concentration of 1.0 ⁇ g / ml.
  • Untreated Jurkat cells and the recombinant Jurkat cells were inoculated into six-well plates, and after the cells were grown, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to amplify the expected genes, and electrophoretic recovery was performed. PCR products.
  • the PCR product was digested with T7 endonuclease I at 37 ° C for 1 h, and then subjected to agarose gel electrophoresis. The results are shown in Figure 1. Compared with the control group, the experimental group showed two distinct cutting bands. This shows that the specific gRNA sequence of the targeted knockout human SLEB2 gene can guide the CRISPR / Cas9 system to successfully edit the human SLEB2 gene.
  • the CRISPR / Cas9 targeted knockout human SLEB2 gene and its specific gRNA provided by the present invention provide an experimental technology platform for in-depth exploration of the role of the SLEB2 gene, and can be used in research and development of drugs related to abnormal expression of the SLEB2 gene.

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Abstract

涉及基于CRISPR/Cas9系统的导向RNA(gRNA)序列及其组合在用于特异性靶向敲除人SLEB2基因的gRNA。根据CRISPR/Cas9的设计原则设计了gRNA，其序列如SEQ ID NO.1所示，并且将其构建在px459载体上。在Jurkat细胞中利用这条gRNA及其组合指导的CRISPR/Cas9系统，可以敲除人SLEB2基因。利用制备的gRNA能够靶向人SLEB2基因并且实现基因敲除。

Description

CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA 技术领域
本发明属于基因工程和基因编辑技术领域，具体涉及CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA。
 背景技术
SLEB2是一种重要的免疫抑制分子，为CD28超家族成员。SLEB2与及其配体PDCD1L1组成的信号通路是机体免疫反应中最重要的信号通路之一，其激活可导致免疫抑制性肿瘤微环境形成，使肿瘤细胞逃避机体免疫监视和杀伤，而阻断SLEB2/ PDCD1L1信号通路可以逆转肿瘤免疫微环境，增强内源性抗肿瘤免疫效应。因此，对SLEB2在肿瘤治疗中所起的作用及其机制的研究非常重要，需要进一步深入研究。
 技术问题
以SLEB2为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义，其潜在的临床转化价值很大，需进行扎实的研究方可投入实际应用，但现有技术中缺乏敲除SLEB2基因表达的手段，对相关研究的进展造成了一定的阻碍。
CRISPR/Cas9基因编辑系统已经成功应用于动植物、细菌、酵母等细胞，是目前最先进的基因组编辑系统，可快速、简便、高效、特异性靶向敲除基因，为高效靶向敲除SLEB2基因，实现SLEB2基因相关疾病的治疗提供了一种可能的选择。本发明的目的就是要验证利用CRISPR/Cas9高效靶向敲除SLEB2基因，提供相应的技术方案，达到特异性敲除SLEB2基因的目的。
 技术解决方案
本发明的目的在于通过设计、构建、筛选，最终提供基于CRISPR/Cas9系统，同时靶向人SLEB2基因的高效gRNA及其靶位点序列，并抑制人SLEB2基因的表达。
       本申请的具体技术方案如下：
       1、靶向人SLEB2基因的高效gRNA及其靶点序列的设计及gRNA/Cas9表达系统构建；
2、在Jurkat细胞模型中，分析检测gRNA指导的CRISPR系统对于人靶位点敲除效率。
 有益效果
本发明提供的CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA为深入探索SLEB2基因的作用提供实验技术平台，可用于与SLEB2基因表达异常相关的药物研究和开发中。
 附图说明
图1为T7核酸内切酶试验结果。
 本发明的实施方式
大肠杆菌Stbl3购自北京全式金，T4 DNA连接酶购自Thermo，px459质粒购自Addgene，Bbs I内切酶购自Fermentas，Lipofectamine 3000和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，基因组DNA提取试剂盒购自北京天根，T7核酸内切酶I购自NEB，PrimeSTAR HS（premix）购自大连宝生物。
实施例一 靶向人 SLEB2 基因的 gRNA 合成
在Genebank中找到人SLEB2基因的序列，并在其CDS区域设计潜在靶位点。通过在线设计工具（http://crispr-era.stanford.edu/）及gRNA的设计原则，评估人SLEB2基因序列上得分较高的gRNA。
在所述gRNA的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，委托合成。
实施例二 px459-SLEB2 载体的构建
将合成的核苷酸序列各自用去离子菌水配成100 μmol/l，放入500 ml 沸水中，自然室温冷却退火，形成双链gRNA序列。
BBs I双酶切px459 质粒，回收后将其与所述gRNA序列按1:6混合后，T4 DNA连接酶16℃连接过夜。转化大肠杆菌Stbl3，氨苄青霉素筛选培养并挑单克隆菌株，测序鉴定。
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100μg/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-SLEB2质粒。
实施例四 Jurkat 细胞转导和筛选
转染前一天将Jurkat细胞接种至六孔板，每孔50万个细胞，培养至第二天，使用Lipofectamine3000将2.5 μg px459-SLEB2重组载体转导至所述Jurkat细胞。培养24 h后，将经转导的细胞消化至60 mm培养皿中，并向所述培养皿中加入含有终浓度为1.0 μg/ml嘌呤霉素进行培养，设为实验组；同时设置未经转导的Jurkat作为对照组，置于含有终浓度为1.0 μg/ml嘌呤霉素的培养基中进行培养。培养7 d后，所述对照组细胞已经全部死亡，实验组出现抗性克隆，然后将实验组的培养基更换为含有终浓度为0.5 μg/ml嘌呤霉素的培养基中继续扩大培养，获得所述重组Jurkat细胞。
实施例五 T7 核酸内切酶试验检测转导效果
分别取未经处理的Jurkat细胞和所述重组Jurkat细胞接种至六孔板，待细胞长满后，提取基因组DNA，然后应用高保真PCR酶PrimeSTAR HS扩增基因编辑预期发生的位点，电泳回收PCR产物。
PCR产物用T7核酸内切酶I在37℃酶切1 h，然后进行琼脂糖凝胶电泳，结果如图1所示，与对照组相比，实验组出现了2条明显的切割条带，说明所述靶向敲除人SLEB2基因的特异性gRNA序列可以引导CRISPR/Cas9系统成功对人SLEB2基因进行编辑。
 工业实用性
本发明提供的CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA为深入探索SLEB2基因的作用提供实验技术平台，可用于与SLEB2基因表达异常相关的药物研究和开发中。

Claims (3)

  1. 在CRISPR/Cas9特异性敲除人SLEB2基因的gRNA，所述gRNA在人SLEB2基因上的靶序列是唯一的，其特征在于：所述gRNA在人SLEB2基因的靶向位点位于人SLEB2基因的外显子上。
  2. 根据权利要求1所述的在CRISPR/Cas9特异性敲除人SLEB2基因的gRNA，其特征在于：其对应的核酸序列如序列SEQ ID NO.1所示。
  3. CRISPR/Cas9特异性敲除人SLEB2基因的gRNA的方法，具体涉及如下步骤：
    （1）如权利要求1-2任意一项所述的gRNA，在其对应DNA序列的5’末端加上CACC得到正向寡核苷酸序列，在其互补链的5’末端加上AAAC得到反向寡核苷酸序列，分别合成正向和反向寡核苷酸序列，然后将合成的序列变性、退火，得到具有Bbs I粘性末端的双链DNA片段；
    （2）将步骤（1）中合成的双链DNA片段和用Bbs I酶切过的px459载体进行连接，将连接产物转化到大肠杆菌Stbl3中，涂布于带有100 μg/ml氨苄青霉素抗性的LB平板上，筛选阳性菌落，提取阳性菌落质粒进行分析及测序，确定gRNA表达载体构建成功，命名为px459-SLEB2；
    （3）将步骤（2）构建的gRNA载体转染Jurkat细胞，并用以未经处理的Jurkat细胞作为对照组。
PCT/CN2018/091722 2018-06-16 2018-06-16 CRISPR/Cas9靶向敲除人SLEB2基因及其特异性gRNA WO2019237392A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
CN106191062A (zh) * 2016-07-18 2016-12-07 广东华南联合疫苗开发院有限公司 一种tcr‑/pd‑1‑双阴性t细胞及其构建方法
CN106399375A (zh) * 2016-08-31 2017-02-15 南京凯地生物科技有限公司 利用CRISPR/Cas9敲除人PD‑1基因构建靶向CD19CAR‑T细胞的方法
CN106434663A (zh) * 2016-10-12 2017-02-22 遵义医学院 CRISPR/Cas9靶向敲除人ezrin基因增强子关键区的方法及其特异性gRNA
CN106480097A (zh) * 2016-10-13 2017-03-08 南京凯地生物科技有限公司 利用CRISPR/Cas9技术敲除人PD‑1基因构建可靶向MSLN新型CAR‑T细胞的方法及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103820454A (zh) * 2014-03-04 2014-05-28 黄行许 CRISPR-Cas9特异性敲除人PD1基因的方法以及用于特异性靶向PD1基因的sgRNA
CN106191062A (zh) * 2016-07-18 2016-12-07 广东华南联合疫苗开发院有限公司 一种tcr‑/pd‑1‑双阴性t细胞及其构建方法
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