WO2020000460A1 - Procédé d'inactivation ciblée de gène lgals9 humain basé sur crispr/cas9 et arng spécifique associé - Google Patents

Procédé d'inactivation ciblée de gène lgals9 humain basé sur crispr/cas9 et arng spécifique associé Download PDF

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Publication number
WO2020000460A1
WO2020000460A1 PCT/CN2018/093870 CN2018093870W WO2020000460A1 WO 2020000460 A1 WO2020000460 A1 WO 2020000460A1 CN 2018093870 W CN2018093870 W CN 2018093870W WO 2020000460 A1 WO2020000460 A1 WO 2020000460A1
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grna
human
lgals9
crispr
sequence
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PCT/CN2018/093870
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English (en)
Chinese (zh)
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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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Priority to PCT/CN2018/093870 priority Critical patent/WO2020000460A1/fr
Publication of WO2020000460A1 publication Critical patent/WO2020000460A1/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 belongs to the field of molecular biology, and particularly relates to a CRISPR / Cas9 targeted knockout method of human Lgals9 gene and a specific gRNA thereof.
  • Lgals9 is a new galectin family protein isolated from mouse embryonic kidney tissue. It is widely distributed in human islet cells, lungs, liver, tonsils, and various immune cells. It is not only distributed in the nucleus, but also in the immune system. In the cytoplasm and even the extracellular matrix.
  • Lgals9 plays an important role in chemotactic eosinophils, mediates cell adhesion, differentiation, apoptosis, cell aggregation, inflammatory response, and tumor metastasis. It can be used for immunotherapy of tumors and requires a lot of research to achieve clinical transformation. . However, the lack of a means to target the expression of Lgals9 gene in the prior art has caused a certain obstacle to the progress of related research.
  • the purpose of the present invention is to provide a method for targeting knockout of human Lgals9 gene and its specific gRNA through design, construction and detection.
  • the present invention uses the CRISPR / Cas9 system principle and gRNA design principles as the basis, and uses software to design gRNA to target the human Lgals9 gene.
  • the corresponding forward and reverse complementary oligonucleotide strands of the synthesized gRNA were annealed to form a double strand, which was connected to the px459 vector to construct a recombinant plasmid; the recombinant plasmid was transfected into Jurkat cells for target site knockout verification.
  • the specificity of the gRNA-guided CRISPR / Cas9 system of the present invention for targeted knockout of human Lgals9 gene was analyzed and identified in a Jurkat cell model.
  • a CRISPR / Cas9 targeted knockout method of human Lgals9 gene and its specific gRNA A CRISPR / Cas9 targeted knockout method of human Lgals9 gene and its specific gRNA:
  • the target sequence of the gRNA in the human Lgals9 gene conforms to the arrangement rule of the 5′-N (20) -NGG-3 ′ or 5′-CCN-N (20) -3 ′ sequence;
  • the gRNA is unique in the target sequence of the human Lgals9 gene.
  • a method for CRISPR / Cas9 targeted knockout of human Lgals9 gene includes the following steps:
  • the px459 vector was digested with BbsI and ligated with the double-stranded gRNA oligonucleotide chain.
  • the ligation product was transformed into E. coli Stbl3 competent cells, and positive clones were selected on ampicillin-resistant plates, followed by sequencing and identification after expansion. ;
  • Lipofectamine 2000 transfection reagent was used to transfect plasmid px459-gRNA into Jurkat cells to achieve targeted knockout of human Lgals9 gene. Then the genomic DNA of the cells was extracted for PCR amplification, and the PCR products were digested with T7 endonuclease I, and the results were confirmed by electrophoresis.
  • the gRNA provided by the present invention can achieve specific knockout of the human Lgals9 gene, and is of great significance for studying the clinical treatment of human Lgals9 gene-related diseases.
  • FIG. 1 is a graph of T7 endonuclease I test results of cells in an experimental group and a control group.
  • the sequence of human Lgals9 gene was found in GenBank, and gRNA was designed using online software. Add a G to the 5 'end of the forward oligonucleotide strand of the gRNA, and a C to the 3' end of the reverse oligonucleotide strand accordingly. At the same time, CACC was added to the 5 ′ end of the forward oligonucleotide strand of each pair of complementary sequences, and AAAC was added to the 5 ′ end of the reverse oligonucleotide strand. The ends formed after annealing were digested with the px459 vector and digested with Bbs I. The sticky ends formed later are complementary.
  • the oligonucleotide chain was diluted to a final concentration of 100 ⁇ mol / L, and an annealing reaction was performed.
  • the reaction system is as follows: two complementary Oligo DNAs 0.5 ⁇ l each, 2 ⁇ l Annealing Buffer (10 ⁇ ), 17 ⁇ l ddH2O. After centrifuging the above system for an instant, place it in a boiling water bath and incubate for 5 min, then take it out and slowly cool it to room temperature for 1-2 hours.
  • the px459 vector was digested with Bbs I enzyme (Fermentas), the target fragment was purified and recovered, and ligated with the gRNA double-stranded DNA to construct a recombinant plasmid px459-gRNA.
  • the ligation reaction system is as follows: 2 ⁇ l gRNA double-stranded DNA, 2 ⁇ l px459 digested fragments, 1 ⁇ l T4 DNA ligase buffer, 1 ⁇ l T4 DNA ligase (NEB), 4 ⁇ l ddH2O. After centrifuging the above system transiently, it was incubated in a 16 ° C water bath overnight. The ligation product was transformed into E.
  • Jurkat cells were seeded on a six-well plate, and 2 ml of 10% FBS-containing DMEM medium was added to each well to cultivate 2 ml. After 18-24 h, the cell confluence rate reached 50% to 60%, which was then used for transfection.
  • the liposome method was used to transfect the recombinant plasmid px459-gRNA according to the instructions of Lipofectamine 2000 transfection reagent.
  • Embodiment 4 T7 Endonuclease I Examine transfection results
  • the transfected Jurkat cells (experimental group) and normal Jurkat cells (control group) were expanded and their genomic DNA was extracted separately, and then amplified by high-fidelity PCR.
  • the PCR product was recovered by electrophoresis, and the product was digested with T7 endonuclease I.
  • the reaction system was: 1 ⁇ l 10 ⁇ T7E1 Buffer, 1 ⁇ l T7 endonuclease I, 1 ⁇ g of the recovered PCR product, made up to 10 with water. ⁇ l.
  • the reaction conditions were: 37 ° C water bath 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 Lgals9 gene in Jurkat cells was successfully edited.
  • the gRNA provided by the present invention can achieve specific knockout of the human Lgals9 gene, and is of great significance for studying the clinical treatment of human Lgals9 gene-related diseases.

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

Abstract

L'invention concerne un procédé d'inactivation ciblée du gène Lgals9 humain basé sur CRISPR/Cas9 et un ARNg spécifique associé.
PCT/CN2018/093870 2018-06-29 2018-06-29 Procédé d'inactivation ciblée de gène lgals9 humain basé sur crispr/cas9 et arng spécifique associé WO2020000460A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093870 WO2020000460A1 (fr) 2018-06-29 2018-06-29 Procédé d'inactivation ciblée de gène lgals9 humain basé sur crispr/cas9 et arng spécifique associé

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093870 WO2020000460A1 (fr) 2018-06-29 2018-06-29 Procédé d'inactivation ciblée de gène lgals9 humain basé sur crispr/cas9 et arng spécifique associé

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WO2020000460A1 true WO2020000460A1 (fr) 2020-01-02

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1744910A (zh) * 2003-01-24 2006-03-08 株式会社嘉尔药物 含有半乳凝素9的医药
CN103623393A (zh) * 2012-08-23 2014-03-12 上海博笛生物科技有限公司 半乳凝素-9在系统性红斑狼疮或类似炎症性疾病中的作用
WO2018024849A1 (fr) * 2016-08-03 2018-02-08 Aalborg Universitet Oligonucléotides antisens (aso) conçus pour inhiber des protéines de points de contrôle immunitaires

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1744910A (zh) * 2003-01-24 2006-03-08 株式会社嘉尔药物 含有半乳凝素9的医药
CN103623393A (zh) * 2012-08-23 2014-03-12 上海博笛生物科技有限公司 半乳凝素-9在系统性红斑狼疮或类似炎症性疾病中的作用
WO2018024849A1 (fr) * 2016-08-03 2018-02-08 Aalborg Universitet Oligonucléotides antisens (aso) conçus pour inhiber des protéines de points de contrôle immunitaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RAN F ET AL.: "Genome Engineering Using the CRISPR-Cas9 System", NATURE PROTOCOLS, vol. 8, no. 11, 1 November 2013 (2013-11-01), pages 2281 - 2306, XP009174668, ISSN: 1754-2189, DOI: 10.1038/nprot.2013.143 *

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