WO2019237391A1 - Inactivation ciblée par crispr/cas9 du gène txgp1 humain et arng spécifique associé - Google Patents

Inactivation ciblée par crispr/cas9 du gène txgp1 humain et arng spécifique associé Download PDF

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Publication number
WO2019237391A1
WO2019237391A1 PCT/CN2018/091721 CN2018091721W WO2019237391A1 WO 2019237391 A1 WO2019237391 A1 WO 2019237391A1 CN 2018091721 W CN2018091721 W CN 2018091721W WO 2019237391 A1 WO2019237391 A1 WO 2019237391A1
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WIPO (PCT)
Prior art keywords
txgp1
grna
gene
human
crispr
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PCT/CN2018/091721
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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/CN2018/091721 priority Critical patent/WO2019237391A1/fr
Publication of WO2019237391A1 publication Critical patent/WO2019237391A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the invention belongs to the technical field of genetic engineering and gene editing, and particularly relates to a CRISPR / Cas9 targeted knockout human TXGP1 gene and a specific gRNA thereof.
  • TXGP1 is a member of the TNF receptor superfamily and is a type I transmembrane glycoprotein. TXGP1 expression profile is limited to the surface of activated CD4 + and CD8 + T cells, and is predominantly CD4 + T cells. Human TXGP1 ligand belongs to the TNF family and is a type II transmembrane glycoprotein. IMD16 / TXGP1 is an important pair of co-stimulatory molecules that play an important role in the body's immune response and various diseases. Their interactions can promote the activation, proliferation, and migration of CD + 4 T cells, extend their life span, and promote germination. The formation of centers and the differentiation of DCs mature.
  • TXGP1 can synergistically stimulate the activation of T cells, promote the production of high titer antibodies and class conversion by B cells, and mediate the infiltration of IMD16 + T cells into the inflammatory response site, which plays an important role in tumor immunotherapy and its potential clinical transformation value It is very large and requires solid research before it can be put into practical use. However, the lack of a means of knocking out TXGP1 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the CRISPR / Cas9 gene editing system has been successfully applied to animals and plants, bacteria, yeast and other cells. It is the most advanced genome editing system currently available. It can quickly, easily, efficiently, and specifically target knockout genes to efficiently knock out TXGP1. Genes, enabling the treatment of TXGP1 gene-related diseases provide a possible option.
  • the purpose of the present invention is to verify the efficient and targeted deletion of TXGP1 gene by using CRISPR / Cas9, provide corresponding technical solutions, and achieve the purpose of specifically deleting TXGP1 gene.
  • the purpose of the present invention is to provide a highly efficient gRNA and its target site sequence based on the CRISPR / Cas9 system, which simultaneously target the human TXGP1 gene, and inhibit the expression of the human TXGP1 gene through design, construction, and screening.
  • the CRISPR / Cas9 targeted knockout human TXGP1 gene and its specific gRNA provided by the present invention provide an experimental technology platform for further exploring the role of the TXGP1 gene, and can be used in research and development of drugs related to abnormal TXGP1 gene expression.
  • FIG 1 shows the results of the T7 endonuclease test
  • Figure 2 shows the Western TXGP1 protein of Jurkat cells in the control and experimental groups Blot test result graph.
  • E. coli Stbl3 was purchased from Beijing Quanshijin, T4 DNA ligase was purchased from Thermo, px459 plasmid was purchased from Addgene, Bbs I endonuclease was purchased from Fermentas, Lipofectamine 3000 and Opti-MEM medium were purchased from Invitrogen, Endo-Free Plasmid Mini Kit was purchased from Omega bio-tek company, genomic DNA extraction kit was purchased from Beijing Tiangen, T7 endonuclease I was purchased From NEB, PrimeSTAR HS (premix) was purchased from Dalian Baobi.
  • the synthesized nucleotide sequences were each made into 100 with deionized bacteria water. ⁇ mol / l, placed in 500 ml of boiling water, and cooled and annealed at room temperature to form a double-stranded gRNA sequence.
  • the correct strain was sequenced and identified in Example 2 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 10,000 rpm at 4 ° C, 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 px459-TXGP1 plasmid without endotoxin.
  • Jurkat cells were seeded into a six-well plate with 500,000 cells per well and cultured until the next day. 2.5 ⁇ g of Lipofectamine 3000 was used.
  • the px459-TXGP1 recombinant vector was transduced into the Jurkat cells. After 24 hours of incubation, the transduced cells were digested into a 60 mm petri dish, and a final concentration of 1.0 ⁇ g / ml puromycin was added to the petri dish for culture, which was set as the experimental group; Transduced Jurkat was used as a control group, and cultured in a medium containing puromycin at a final concentration of 1.0 ⁇ g / ml.
  • Untreated Jurkat cells and the recombinant Jurkat cells were inoculated into six-well plates, and after the cells were grown, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to amplify the expected genes, and electrophoretic recovery was performed. PCR products.
  • the PCR product was digested with T7 endonuclease I at 37 ° C for 1 h, 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 bands. This shows that the specific gRNA sequence of the targeted knockout human TXGP1 gene can guide the CRISPR / Cas9 system to successfully edit the human TXGP1 gene.
  • the antibody was detected using ANTI-TXGP1 mouse monoclonal antibody at a concentration of 1: 1000.
  • the test results are shown in Figure 2. It can be seen that Western Blot did not detect TXGP1 protein bands in the experimental group cells, while TXGP1 protein bands appeared in the control group, indicating the specificity of the targeted knockout human TXGP1 gene.
  • the gRNA sequence can knock out the TXGP1 gene.
  • the CRISPR / Cas9 targeted knockout human TXGP1 gene and its specific gRNA provided by the present invention provide an experimental technology platform for in-depth exploration of the role of TXGP1 gene, and can be used in research and development of drugs related to abnormal expression of TXGP1 gene.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un ARN guide (ARNg) dont la séquence d'ARN est basée sur un système CRISPR/Cas9 et dont une combinaison est utilisée pour l'inactivation ciblée spécifique d'un gène TXGP1 humain. L'ARNg est conçu selon les principes de conception de CRISPR/Cas9 dont la séquence est représentée dans SEQ ID NO. 1, l'ARNg étant construit sur un vecteur px459. Un gène TXGP1 humain peut être inactivé efficacement dans les cellules Jurkat à l'aide d'un système CRISPR/Cas9 guidé par un tel ARN et une combinaison de celui-ci. L'ARNg préparé peut être utilisé pour cibler précisément un gène TXGP1 humain et effectuer une inactivation de gène. Le procédé de préparation est de fonctionnement simple, le ciblage par l'ARNg est bon, et l'efficacité d'inactivation du système CRISPR/Cas9 est élevée.
PCT/CN2018/091721 2018-06-16 2018-06-16 Inactivation ciblée par crispr/cas9 du gène txgp1 humain et arng spécifique associé WO2019237391A1 (fr)

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PCT/CN2018/091721 WO2019237391A1 (fr) 2018-06-16 2018-06-16 Inactivation ciblée par crispr/cas9 du gène txgp1 humain et arng spécifique associé

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PCT/CN2018/091721 WO2019237391A1 (fr) 2018-06-16 2018-06-16 Inactivation ciblée par crispr/cas9 du gène txgp1 humain et arng spécifique associé

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020160489A1 (fr) * 2019-02-01 2020-08-06 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie

Citations (6)

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CN105647922A (zh) * 2016-01-11 2016-06-08 中国人民解放军疾病预防控制所 基于一种新gRNA序列的CRISPR-Cas9系统在制备乙肝治疗药物中的应用
CN105950541A (zh) * 2016-06-21 2016-09-21 中国医学科学院医学生物学研究所 一种hFGF21基因敲除人肝细胞株的构建方法
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
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral
CN107418974A (zh) * 2017-07-28 2017-12-01 新乡医学院 一种利用单克隆细胞分选快速获得CRISPR/Cas9基因敲除稳定细胞株的方法
CN107841509A (zh) * 2017-12-06 2018-03-27 南方医科大学南方医院 一种敲除胶质母细胞瘤dhc2基因的试剂盒

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CN105647922A (zh) * 2016-01-11 2016-06-08 中国人民解放军疾病预防控制所 基于一种新gRNA序列的CRISPR-Cas9系统在制备乙肝治疗药物中的应用
WO2017152149A1 (fr) * 2016-03-03 2017-09-08 University Of Massachusetts Adn double hélice linéaire à extrémité fermée pour transfert de gène non viral
CN105950541A (zh) * 2016-06-21 2016-09-21 中国医学科学院医学生物学研究所 一种hFGF21基因敲除人肝细胞株的构建方法
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
CN107418974A (zh) * 2017-07-28 2017-12-01 新乡医学院 一种利用单克隆细胞分选快速获得CRISPR/Cas9基因敲除稳定细胞株的方法
CN107841509A (zh) * 2017-12-06 2018-03-27 南方医科大学南方医院 一种敲除胶质母细胞瘤dhc2基因的试剂盒

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ZHANG, . CHUNPAN ET AL.: "Study on the Role and Mechanism of OX40 in Acute Liver Failure Induced by D-galactosamine/Lipopolysaccharide", JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 17, no. 7, 30 April 2018 (2018-04-30), pages 673 - 677 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020160489A1 (fr) * 2019-02-01 2020-08-06 KSQ Therapeutics, Inc. Compositions de régulation génique et procédés pour améliorer l'immunothérapie

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