WO2019237386A1 - Procédé d'invalidation du gène ct1.1 humain - Google Patents

Procédé d'invalidation du gène ct1.1 humain Download PDF

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Publication number
WO2019237386A1
WO2019237386A1 PCT/CN2018/091716 CN2018091716W WO2019237386A1 WO 2019237386 A1 WO2019237386 A1 WO 2019237386A1 CN 2018091716 W CN2018091716 W CN 2018091716W WO 2019237386 A1 WO2019237386 A1 WO 2019237386A1
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WIPO (PCT)
Prior art keywords
cells
gene
sgrna
sequence
lentivirus
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PCT/CN2018/091716
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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/CN2018/091716 priority Critical patent/WO2019237386A1/fr
Publication of WO2019237386A1 publication Critical patent/WO2019237386A1/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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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, and in particular, to a method for knocking human CT1.1 gene by using CRISPR-Cas9 gene editing technology.
  • Cancer / testis antigen is a group of tumor-associated antigens. It is not expressed in normal human tissues except testis, placenta, and trophoblast cells, but is highly expressed in a variety of tumor tissues, making it a recent year. Tumor immunotherapeutic target antigen has been widely concerned.
  • Melanin-associated antigen is a class of tumor-associated antigens isolated from melanoma cells. It is a subfamily of CTA. It can be processed into antigen peptides in tumor cells, combined with human leukocyte antigen I, and then Autocytotoxic T lymphocytes recognize and kill tumor cells.
  • CT1.1 is the earliest found member of the MAGE family. It is specifically expressed in malignant tumors and can bind to MHC molecules. It can be recognized by specific cell T-cell receptors and activated into cytotoxic T-lymph with specific T-cell receptors. Cells enable the body to produce an immune response that is dominated by cellular immunity. Therefore, CT1.1 is a tumor-specific ideal target antigen, which has become a hot topic in recent years. However, the lack of a specific method for human CT1.1 gene deletion in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a method for knocking out the human CT1.1 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 up and cultured, and then a large number of plasmids were extracted to obtain a constructed CRISPR-Cas9 containing the knock-out CT1.1 gene System expression plasmid, save for future 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 screened A375 cells (experimental group) and normal A375 cells without any treatment were taken, respectively, and genomic DNA was extracted and used as a template for PCR amplification. After reannealing, treatment with T7E1 enzyme and agarose gel electrophoresis Observe the results of CT1.1 gene knockout.
  • the method for knocking out the CT1.1 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 CT1.1 gene, and can be used in research and development of drugs related to abnormal CT1.1 expression.
  • Figure 1 shows the results of identifying the CT1.1 gene editing status 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’- GAGGTTTCCATTCTGAGGGA -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 ' -CACCGAGGTTTCCATTCTGAGGGA -3 'and 5'-AAACTCCCTCAGAATGGAAACCTC -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-CT1.1 are obtained.
  • Example 3 Packaging of lentivirus
  • Lipofectamine 3000 was co-transfected into 293T cells. 48 hours before transfection, inoculate cells into a well plate or petri dish for lentivirus production. During transfection, the confluence of cells is about 70% -80% is the best infection state, and the viability is ⁇ 95%. The dyeing time was the starting point, and the harvest time was 48 h and 72 h. After filtration with a ⁇ m filter, it was stored at -80 ° C.
  • Embodiment 4 A375 Lentiviral infection of cells and puromycin selection
  • Embodiment 5 T7E1 Enzyme identification CT1.1 Knockout results
  • A375 cells (experimental group) and normal A375 cells (control group) infected with lentivirus were expanded and cultured, and their 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 CT1.1 gene in A375 cells was successfully edited.
  • the method for knocking out the CT1.1 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 CT1.1 gene, and can be used in research and development of drugs related to abnormal CT1.1 expression.

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

Abstract

L'invention concerne un procédé d'invalidation du gène CT1.1 humain au moyen d'une technologie d'édition du gène 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 du plasmide des cellules 293T et leur conditionnement en lentivirus; 4) l'infection lentivirale de la cellule cible et le criblage avec la puromycine; et 5) la vérification du résultat de l'inactivation du gène CT1.1. Le procédé est applicable à la recherche et au développement de médicaments associés à CT1.1.
PCT/CN2018/091716 2018-06-16 2018-06-16 Procédé d'invalidation du gène ct1.1 humain WO2019237386A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091716 WO2019237386A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation du gène ct1.1 humain

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091716 WO2019237386A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation du gène ct1.1 humain

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WO2019237386A1 true WO2019237386A1 (fr) 2019-12-19

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106480080A (zh) * 2012-12-12 2017-03-08 布罗德研究所有限公司 用于改变基因产物的表达的crispr‑cas系统和方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106480080A (zh) * 2012-12-12 2017-03-08 布罗德研究所有限公司 用于改变基因产物的表达的crispr‑cas系统和方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHENG GUAN ET AL: "Knockdown MAG-1 by RNA Interference System in U87 of the Human Glioma Cell line", JOURNAL OF MEDICAL POSTGRADUATES, vol. 19, no. 4, 30 April 2006 (2006-04-30), ISSN: 1008-8199 *
JINGJING WANG ET AL: "Establishment of MAGEC2-knockout cells and functional investigation of MAGEC2 in tumor cells", CANCER SCIENCE, vol. 107, no. 12, 1 December 2016 (2016-12-01), pages 1888 - 1897, XP055673589, ISSN: 1347-9032, DOI: 10.1111/cas.13082 *
MEEK DAVID , MARCAR LYNNETTE: "MAGE-A antigens as targets in tumour therapy", CANCER LETTERS, vol. 324, no. 2, 28 November 2012 (2012-11-28), pages 126 - 132, XP055673596, ISSN: 0304-3835, DOI: 10.1016/j.canlet.2012.05.011 *

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