WO2019237394A1 - Procédé de ciblage de l'invalidation du gène alps5 humain par utilisation du système crispr/cas9 - Google Patents

Procédé de ciblage de l'invalidation du gène alps5 humain par utilisation du système crispr/cas9 Download PDF

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Publication number
WO2019237394A1
WO2019237394A1 PCT/CN2018/091724 CN2018091724W WO2019237394A1 WO 2019237394 A1 WO2019237394 A1 WO 2019237394A1 CN 2018091724 W CN2018091724 W CN 2018091724W WO 2019237394 A1 WO2019237394 A1 WO 2019237394A1
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WIPO (PCT)
Prior art keywords
alps5
human
gene
crispr
knockout
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PCT/CN2018/091724
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English (en)
Chinese (zh)
Inventor
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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Priority to PCT/CN2018/091724 priority Critical patent/WO2019237394A1/fr
Publication of WO2019237394A1 publication Critical patent/WO2019237394A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N15/63—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

Definitions

  • the present invention relates to the field of molecular biology, in particular to a method for targeted knockout of human ALPS5 gene by using CRISPR / Cas9 system.
  • the human anti-cancer immune mechanism includes two aspects of cellular immunity and humoral immunity. However, it is mainly based on cellular immunity.
  • the effector cells that play a major role in the cellular immune mechanism are T cells, of which CD8 + T cells are the most important effect in tumor immunity. Perform the cell.
  • T-cell activation requires dual signal recognition.
  • the tumor antigen first binds to MHC molecules on antigen-presenting cells or target cells, and then binds to T-lymphocyte surface antigen recognition receptors to provide the first signal for T-cell activation; CD28 on the T-cell surface Binding to B7 molecules on the surface of antigen presenting cells or on the surface of target cells provides a second signal.
  • the T cells are fully activated, and the activated T cells first proliferate, generating a large number of antigen-specific T cells, which migrate to the tumor site to play a killing role.
  • negative regulatory molecules such as ALPS5 began to be upregulated.
  • ALPS5 is a transmembrane protein and belongs to the CD28 family.
  • the ligand of ALPS5 is a member of the B7 family. It competes with CD28 molecules and binds to B7-1 and B7-2. It inhibits the proliferation of activated T cells and negatively regulates immunity. Under physiological conditions, it can prevent the immune from being enlarged. It maintains a dynamic balance between health and disease, but is often used by tumor cells to achieve the purpose of immune escape. Therefore, the research on the role of ALPS5 in tumorigenesis and development can promote the development of tumor therapy. However, the lack of targeted knockout of ALPS5 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a method for applying the CRISPR / Cas9 system to target the knockout of human ALPS5 gene.
  • the present invention is implemented as follows:
  • a gRNA targeting ALPS5 is designed;
  • the px459 plasmid was digested by Bbs I, and the digested product was recovered by electrophoresis;
  • reaction temperature of the BBs I digestion system was 37 ° C.
  • time was 2 hours
  • digestion system was: 2 ⁇ g of px459 plasmid, 3 ⁇ l of 10 ⁇ FastDigest Buffer, 3 ⁇ l of Bbs I, and made up to 30 ⁇ l with water.
  • the liposome is Lipofectamine 2000.
  • the recipient cells are selected from Jurkat cells.
  • the T7 Endonuclease I reaction system is: 1 ⁇ l 10 ⁇ T7E1 Buffer, 1 ⁇ l T7 Endonuclease I, 1 ⁇ g of recovered PCR product, made up to 10 ⁇ l with water.
  • the reaction conditions were: 37 ° C water bath for 1 h.
  • the above-mentioned method using the CRISPR / Cas9 system to target human ALPS5 gene knockout can be used for human ALPS5 gene knockout.
  • the method for applying the CRISPR / Cas9 system to target the knockout of human ALPS5 gene provided by the present invention provides an experimental technology platform for further exploring the role of ALPS5 gene, and can be used in the research and development of drugs related to abnormal expression of ALPS5 gene.
  • FIG. 1 shows the T7 Endonuclease of Jurkat cells in the control and experimental groups. I test results chart.
  • a gRNA targeting ALPS5 was designed, and its upstream sequence was 5’-ACCGATAAAGTCCTTGATTCTGTG -3 ', and the downstream sequence is 5'-AAACCACAGAATCAAGGACTTTAT-3'. After dissolving, 5 ⁇ l of each was mixed, heated at 95 ° C. for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with Bbs I sticky ends.
  • the px459 plasmid was digested with Bbs I, and the digested product was subjected to agarose gel electrophoresis. The gel was recovered and the concentration was determined.
  • the correct strain was sequenced and identified in Example 1, 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 endotoxin-free pxALPS5 plasmid.
  • Jurkat cells were seeded in a 35 mm petri dish with a seeding density of 50% and DMEM containing 10% fetal bovine serum (FBS) under 37 ° C and 5% CO2.
  • FBS fetal bovine serum
  • 2 ⁇ g of pxALPS5 plasmid was introduced according to the instructions of the Lipofectamine 2000 kit.
  • 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.
  • Untreated Jurkat cells and Jurkat cells introduced with pxALPS5 plasmid were inoculated into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to amplify the expected sites of gene editing. Electrophoresis The PCR product was recovered.
  • T7 Endonuclease I was digested for 1 h at 37 ° C 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 strips. The band indicates that the gRNA sequence targeting the ALPS5 gene guided the CRISPR / Cas9 system to successfully edit the human ALPS5 gene.
  • the method for applying the CRISPR / Cas9 system to target the knockout of human ALPS5 gene provided by the present invention provides an experimental technology platform for further exploring the role of ALPS5 gene, and can be used in the research and development of drugs related to abnormal expression of ALPS5 gene.

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  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne un procédé pour le ciblage de l'invalidation du gène ALPS5 humain par utilisation du système CRISPR/Cas9. Le procédé comprend : la conception d'un ARNg qui cible le gène ALPS5 humain conformément au principe de conception PAM de l'ARNg ; la liaison de l'ARNg au vecteur px459 pour obtenir un vecteur recombinant pour le ciblage de l'invalidation du gène ALPS5 humain ; la transformation du vecteur recombinant en des cellules Jurkat ; et la mise en œuvre du ciblage de l'invalidation du gène ALPS5 humain.
PCT/CN2018/091724 2018-06-16 2018-06-16 Procédé de ciblage de l'invalidation du gène alps5 humain par utilisation du système crispr/cas9 WO2019237394A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091724 WO2019237394A1 (fr) 2018-06-16 2018-06-16 Procédé de ciblage de l'invalidation du gène alps5 humain par utilisation du système crispr/cas9

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091724 WO2019237394A1 (fr) 2018-06-16 2018-06-16 Procédé de ciblage de l'invalidation du gène alps5 humain par utilisation du système crispr/cas9

Publications (1)

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WO2019237394A1 true WO2019237394A1 (fr) 2019-12-19

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
CN106995821A (zh) * 2016-01-26 2017-08-01 深圳市儿童医院 Jurkat-KI-R5细胞系及其构建方法和应用
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用
CN107847524A (zh) * 2015-03-27 2018-03-27 哈佛学院校长同事会 经过修饰的t细胞及其制备和使用方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107847524A (zh) * 2015-03-27 2018-03-27 哈佛学院校长同事会 经过修饰的t细胞及其制备和使用方法
CN106995821A (zh) * 2016-01-26 2017-08-01 深圳市儿童医院 Jurkat-KI-R5细胞系及其构建方法和应用
CN107815468A (zh) * 2016-08-31 2018-03-20 北京百奥赛图基因生物技术有限公司 人源化基因改造动物模型的制备方法及应用
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

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