WO2020000437A1 - Procédé d'invalidation du gène aprf humain - Google Patents

Procédé d'invalidation du gène aprf humain Download PDF

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Publication number
WO2020000437A1
WO2020000437A1 PCT/CN2018/093841 CN2018093841W WO2020000437A1 WO 2020000437 A1 WO2020000437 A1 WO 2020000437A1 CN 2018093841 W CN2018093841 W CN 2018093841W WO 2020000437 A1 WO2020000437 A1 WO 2020000437A1
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WIPO (PCT)
Prior art keywords
cells
aprf
gene
sgrna
lentivirus
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Application number
PCT/CN2018/093841
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English (en)
Chinese (zh)
Inventor
毛吉炎
Original Assignee
深圳市博奥康生物科技有限公司
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Publication date
Application filed by 深圳市博奥康生物科技有限公司 filed Critical 深圳市博奥康生物科技有限公司
Priority to PCT/CN2018/093841 priority Critical patent/WO2020000437A1/fr
Publication of WO2020000437A1 publication Critical patent/WO2020000437A1/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
    • C12N15/86—Viral vectors

Definitions

  • the invention mainly relates to the field of genetic engineering, in particular to a method for knocking human APRF genes by using CRISPR-Cas9 gene editing technology.
  • STAT transcriptional activators
  • APRF is an important member of the STAT family and is closely related to cell proliferation, apoptosis and differentiation. Under normal conditions, the activation of APRF by extracellular signals is strictly regulated. The process of APRF activation is short and rapid, which can maintain the biological function of normal cells. Deletion of APRF causes death of early embryonic cells.
  • APRF is continuously activated.
  • Continuous activation of APRF can mediate tumor immune escape, promote tumor neovascularization, inhibit tumor cell apoptosis, promote tumor cell proliferation and other mechanisms, leading to malignant transformation of tumor cells. Therefore, APRF is an ideal target for the treatment of potential tumors, but the lack of a method for specifically knocking out human APRF genes in the prior art has caused a certain obstacle to the progress of related research.
  • the present invention provides a method for knocking out human APRF gene by using CRISPR-Cas9 gene editing technology.
  • the specific operation steps are as follows:
  • nt sequence is used as the sgRNA to be selected to ensure that it has no homology or low homology with the sequences of other genes, and its sequence is shown in SEQ ID NO.1.
  • SEQ ID NO. 2 and SEQ ID NO. 3 respectively. Entrust the company to synthesize these two sequences;
  • dsDNA Dilute 2 synthetic single-stranded sgRNA sequences to After 100 ⁇ mol / L, dsDNA was mixed and annealed to form dsDNA, and then ligated to the lenti CRISPR v2 vector treated with BsmBI endonuclease.
  • the above products were transformed into E. coli competent cells Stbl3 according to the conventional molecular cloning technology method, and positive clones were selected. The positive clones were picked and expanded and cultured. A large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 system containing the knockout APRF gene. Expression plasmid, save for later use;
  • lentivirus and culture medium containing 4 ⁇ g / mL polybrene
  • the lentiviral solution was changed to a complete medium containing 1 ⁇ g / mL puromycin, and the screening culture was started. 7-14 After d, the cells infected with lentivirus will form single cell clones, and the cell selection is completed.
  • the selected HepG2 cells (experimental group) and normal HepG2 cells without any treatment were extracted respectively, and their genomic DNA was extracted and used as a template for PCR amplification. After re-annealing, they were treated with T7E1 enzyme and agarose gel electrophoresis Observe the results of APRF gene knockout.
  • the method for knocking out the APRF gene provided by the present invention and the cell strain constructed by applying the method provide an experimental technology platform for further exploring the role of the APRF gene, and can be used in research and development of drugs related to abnormal expression of APRF.
  • FIG. 1 is a graph of the results of identifying the editing status of the APRF gene by the T7E1 enzyme.
  • Embodiment one sgRNA the design of
  • nt sequence is used as the sgRNA to be selected to ensure that it has no homology or low homology with the sequences of other genes. Its sequence is 5’- GAAGGGCCTCTCCGAGCCGA -3 ’, such as SEQ ID NO.1. According to the actual needs, the two strands of sgRNA need to be synthesized separately: the CACC sequence needs to be added to the 5 'end of the sgRNA sense strand, and the AAAC sequence needs to be added to the 5' end of the sgRNA antisense strand for subsequent connection.
  • sequences of the two strands are 5 ' -CACCGAAGGGCCTCTCCGAGCCGA -3 'and 5'- AAACTCGGCTCGGAGAGGCCCTTC -3', such as SEQ ID NO. 2 and SEQ ID NO. 3 are shown.
  • the company was commissioned to synthesize the two sequences.
  • the synthesized two single-stranded sgRNA sequences were diluted to 100 ⁇ mol / L, mixed in equal amounts and annealed to form dsDNA, and then ligated to the lenti CRISPR v2 vector treated with BsmBI endonuclease.
  • the above products were transformed into E. coli competent cells Stbl3 according to the conventional molecular cloning technology method, and positive clones were selected.
  • the positive clones were picked up and cultured, and then verified by sequencing to screen out positive clones E. coli containing sequences that fully matched the expected. It performs expansion culture, and then uses the endotoxin-free plasmid extraction kit to extract the recombinant vector therein, and a large number of constructed CRISPR-Cas9 system-containing expression vectors pLentiCRISPR-APRF are obtained.
  • Example 3 Packaging of lentivirus
  • Embodiment 4 HepG2 Lentiviral infection of cells and puromycin selection
  • Embodiment 5 T7E1 Enzyme identification APRF Knockout results
  • the lentivirus-infected HepG2 cells (experimental group) and normal HepG2 cells (control group) were expanded and cultured. Genomic DNA was extracted and amplified by high-fidelity PCR. The PCR product was recovered by electrophoresis, and then the product was digested with T7 endonuclease I at 37 ° C for 1 h. After the digestion, 1% agarose gel electrophoresis was performed, and the results are shown in FIG. 1. It can be seen that the PCR product of the control group was still only one band after digestion, while the experimental group showed multiple bands, indicating that the APRF gene in HepG2 cells was successfully edited.
  • the method for knocking out the APRF gene provided by the present invention and the cell strain constructed by applying the method provide an experimental technology platform for further exploring the role of the APRF gene, and can be used in research and development of drugs related to abnormal expression of APRF.

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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical 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)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne un procédé d'invalidation du gène APRF humain en faisant appel à la technologie d'édition de gènes CRISPR-Cas9. Le procédé comprend les étapes fonctionnelles spécifiques suivantes : (1) la conception d'une séquence d'ARNsg ; (2) la ligature, la transformation et l'amplification de l'ARNsg ; (3) la transfection des plasmides des cellules 293T et leur conditionnement en lentivirus ; (4) l'infection lentivirale de la cellule cible et son dépistage avec la puromycine ; et (5) la vérification du résultat de l'invalidation du gène APRF.
PCT/CN2018/093841 2018-06-29 2018-06-29 Procédé d'invalidation du gène aprf humain WO2020000437A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093841 WO2020000437A1 (fr) 2018-06-29 2018-06-29 Procédé d'invalidation du gène aprf humain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093841 WO2020000437A1 (fr) 2018-06-29 2018-06-29 Procédé d'invalidation du gène aprf humain

Publications (1)

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WO2020000437A1 true WO2020000437A1 (fr) 2020-01-02

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574524A (zh) * 2022-03-14 2022-06-03 深圳市体内生物医药科技有限公司 一种从全基因组中筛选肝癌抑癌基因的方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1113517A (zh) * 1994-04-04 1995-12-20 岸本忠三 转录因子aprf

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1113517A (zh) * 1994-04-04 1995-12-20 岸本忠三 转录因子aprf

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAN, F.A. ET AL.: "Genome Engineering Using the CRISPR-Cas9 System", NATURE PROTOCOLS, vol. 8, no. 11, 24 October 2013 (2013-10-24), pages 2281 - 2306, XP009174668, ISSN: 1754-2189 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114574524A (zh) * 2022-03-14 2022-06-03 深圳市体内生物医药科技有限公司 一种从全基因组中筛选肝癌抑癌基因的方法及其应用

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