WO2019237390A1 - Procédé d'inactivation d'un gène hdmx humain - Google Patents

Procédé d'inactivation d'un gène hdmx humain Download PDF

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Publication number
WO2019237390A1
WO2019237390A1 PCT/CN2018/091720 CN2018091720W WO2019237390A1 WO 2019237390 A1 WO2019237390 A1 WO 2019237390A1 CN 2018091720 W CN2018091720 W CN 2018091720W WO 2019237390 A1 WO2019237390 A1 WO 2019237390A1
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WIPO (PCT)
Prior art keywords
hdmx
cells
gene
sgrna
lentivirus
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PCT/CN2018/091720
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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/CN2018/091720 priority Critical patent/WO2019237390A1/fr
Publication of WO2019237390A1 publication Critical patent/WO2019237390A1/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
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention mainly relates to the field of genetic engineering, in particular to a method for knocking human HDMX genes by using CRISPR-Cas9 gene editing technology.
  • the p53 tumor suppressor gene is located on the short arm of human chromosome 17 and encodes a protein containing 393 amino acids, namely the p53 protein.
  • Wild-type p53 protein can be used as a cell cycle regulator protein to monitor damage and induce apoptosis, inhibit excessive cell proliferation, and have anti-tumor cell proliferation functions.
  • a variety of target genes of the p53 gene mainly include: P21, HDMX, bax, etc. It is these boot genes regulated by the p53 gene and form a network to achieve the functions jointly. Among them, HDMX-p53-p21 signaling pathway is one of the important pathways in the gene pathway.
  • the human HDMX gene encodes a protein with a molecular weight of kD.
  • P1 is upstream of the coding gene
  • P2 is in the first intron, which is controlled by p53 through two nearby p53 binding sites.
  • HDMX-encoded protein is divided into 4 functional regions: about 10 amino acid residues at the N-terminus, which can be combined with p53. This region also has a nuclear localization sequence and a highly acidic region of nuclear export signals; a zinc finger structure that can bind to genes and activate genes To make cells from G1 phase to S phase; ring finger structure, which can mediate protein-protein interactions, participate in cell regulation and promote cell proliferation.
  • HDMX negatively regulates p53 through multiple pathways.
  • HDMX protein can directly bind to p53 to inhibit its activity, resulting in p53 being degraded by the ubiquitin system.
  • Different splicing forms of HDMX also participate in regulatory activity.
  • the abnormal amplification of HDMX or the increase in protein expression levels lead to the inactivation of p53 function, so it is considered to be a newly discovered important proto-oncogene.
  • HDMX protein has been confirmed to be closely related to the degree of tumor metastasis. In tumor metastasis and recurrence, high amplification of HDMX gene or high expression of HDMX protein were observed. Therefore, HDMX is an ideal target for potential tumor treatment.
  • the lack of a method for specifically knocking out the human HDMX gene 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 HDMX 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, such as SEQ ID NO.1.
  • SEQ ID NO. 2 and SEQ ID NO. 3 respectively. Entrust the company to synthesize these two sequences;
  • 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 HDMX 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.
  • the cells infected with lentivirus will form single cell clones, and the cell selection is completed.
  • the HDMX gene knockout method provided by the present invention and a cell line constructed by using the method provide an experimental technology platform for further exploring the role of the HDMX gene, and can be used in research and development of drugs related to abnormal HDMX expression.
  • Figure 1 shows the results of identification of the editing status of HDMX genes 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’- GTGAAACTGTTAGAGCCTTT -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 ' - CACCGTGAAACTGTTAGAGCCTTT -3 ’and 5’- AAACAAAGGCTCTAACAGTTTCAC -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 is used for expansion culture, and then the endotoxin-free plasmid extraction kit is used to extract the recombinant vector therein, and a large number of constructed CRISPR-Cas9 system-containing expression vectors pLentiCRISPR-HDMX are obtained.
  • Example 3 Packaging of lentivirus
  • the 293T cells were thawed and cultured, and the cells were transfected twice after growth and culture.
  • Embodiment 4 MCF-7 Lentiviral infection of cells and puromycin selection
  • Culture MCF-7 cells When the confluency of the cells is about 70% -80%, add a mixture of lentivirus and culture medium (containing 4 ⁇ g / mL polybrene) and treat 24 After h, the lentiviral solution was changed to a complete medium containing 1 ⁇ g / mL puromycin, and the screening culture was started. The screening time was 7-14 days. Change the fluid every other day. Cells infected with lentivirus will form single-cell clones, and the cell selection is complete.
  • lentivirus and culture medium containing 4 ⁇ g / mL polybrene
  • Embodiment 5 T7E1 Enzyme identification HDMX Knockout results
  • the MCF-7 cells infected with lentivirus (experimental group) and normal MCF-7 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 HDMX gene in MCF-7 cells was successfully edited.
  • the HDMX gene knockout method provided by the present invention and a cell line constructed by using the method provide an experimental technology platform for further exploring the role of the HDMX gene, and can be used in research and development of drugs related to abnormal HDMX expression.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé d'inactivation d'un gène HDMX humain à l'aide d'une technologie d'édition de gène CRISPR-Cas9, comprenant : (1) la conception d'une séquence d'ARNsg ; (2) la ligature, la transformation et l'amplification de l'ARNsg ; (3) la transfection d'une cellule 293T avec un plasmide et l'encapsulation dans un lentivirus ; (4) l'infection d'une cellule cible avec le lentivirus et le criblage à l'aide de puromycine ; (5) la vérification d'un résultat d'inactivation de gène HDMX. Dans le procédé, un gène HDMX d'une cellule peut être inactivé et peut être utilisé pour la recherche et le développement de médicaments associés à HDMX.
PCT/CN2018/091720 2018-06-16 2018-06-16 Procédé d'inactivation d'un gène hdmx humain WO2019237390A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091720 WO2019237390A1 (fr) 2018-06-16 2018-06-16 Procédé d'inactivation d'un gène hdmx humain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091720 WO2019237390A1 (fr) 2018-06-16 2018-06-16 Procédé d'inactivation d'un gène hdmx humain

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107746845A (zh) * 2016-12-28 2018-03-02 北京微旋基因技术有限公司 特异性靶向LAG‑3基因的sgRNA和特异性敲除LAG‑3基因的方法
CN107760680A (zh) * 2016-12-28 2018-03-06 北京微旋基因技术有限公司 特异性靶向TIM‑3基因的sgRNA和特异性敲除TIM‑3基因的方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107746845A (zh) * 2016-12-28 2018-03-02 北京微旋基因技术有限公司 特异性靶向LAG‑3基因的sgRNA和特异性敲除LAG‑3基因的方法
CN107760680A (zh) * 2016-12-28 2018-03-06 北京微旋基因技术有限公司 特异性靶向TIM‑3基因的sgRNA和特异性敲除TIM‑3基因的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GAO XIN: "Knocking out Human HOIP Gene by CRISPR/cas9 lentiviral System", SCIENCE TECHNOLOGY AND ENGINEERING, vol. 16, no. 20, 31 July 2016 (2016-07-31), pages 26 - 29 *
SU FENGJU: "Investigation of HDM4 and It's Transcript variants in Tumor Regulation", WANFANG DATA KNOWLEDGE SERVICE PLATFORM, 30 November 2011 (2011-11-30) *
ZHANG YAN: "Effect of Knockout Integrin Gene on Invasion and metastasis of Mouse Breast Cancer cells", CHINESE MASTER'S THESES FULL-TEXT DATABASE , MEDICAL AND HEALTH SCIENCES, no. 1, 15 January 2018 (2018-01-15), pages 12 - 14, ISSN: 1674-024 *

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