WO2019237396A1 - Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9 - Google Patents

Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9 Download PDF

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Publication number
WO2019237396A1
WO2019237396A1 PCT/CN2018/091726 CN2018091726W WO2019237396A1 WO 2019237396 A1 WO2019237396 A1 WO 2019237396A1 CN 2018091726 W CN2018091726 W CN 2018091726W WO 2019237396 A1 WO2019237396 A1 WO 2019237396A1
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Prior art keywords
tnfsf18
human
gene
knockout
crispr
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Ceased
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PCT/CN2018/091726
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English (en)
Chinese (zh)
Inventor
毛吉炎
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Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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Priority to PCT/CN2018/091726 priority Critical patent/WO2019237396A1/fr
Publication of WO2019237396A1 publication Critical patent/WO2019237396A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Definitions

  • the invention relates to the field of molecular biology, in particular to a method for targeted knockout of the human TNFSF18 gene by using a CRISPR / Cas9 system.
  • TNFSF18 is a ligand for the surface molecule CD357 on CD4 + CD25 + Treg cells of thymus origin. Studies have shown that CD357 / TNFSF18 has many important biological activities, including cell proliferation, differentiation, and survival.
  • the CD357 / TNFSF18 system participates in the role of Treg cells in immune regulation, plays an important role in tumor immunotherapy, and has good clinical transformation prospects, but the prior art lacks a means to target the knockout of TNFSF18 gene expression. Related research Progress has caused certain obstacles.
  • the present invention provides a method for applying the CRISPR / Cas9 system to target the knockout of human TNFSF18 gene.
  • the present invention is implemented as follows:
  • a method of using the CRISPR / Cas9 system to target the knockout of human TNFSF18 gene is as follows:
  • a gRNA targeting TNFSF18 is designed;
  • the px459 plasmid was digested by Bbs I, and the digested product was recovered by electrophoresis;
  • Liposomes transduce pxTNFSF18 into the recipient cells.
  • the recipient cells were screened for 7 days at a working concentration of puromycin to expand the recipient cells and extract genomic DNA.
  • the PCR products were recovered by electrophoresis after PCR amplification, and then T7 Endonuclease was used. I Digest the product and identify it by electrophoresis.
  • reaction temperature of the BBs I digestion system was 37 ° C.
  • time was 2 hours
  • digestion system was: 2 ⁇ g of px459 plasmid, 3 ⁇ l of 10 ⁇ FastDigest Buffer, 3 ⁇ l of Bbs I, and made up to 30 ⁇ l with water.
  • the liposome is Lipofectamine 2000.
  • the recipient cells are selected from Jurkat cells.
  • the T7 Endonuclease I reaction system is: 1 ⁇ l 10 ⁇ T7E1 Buffer, 1 ⁇ l T7 Endonuclease I, 1 ⁇ g of recovered PCR product, made up to 10 ⁇ l with water.
  • the reaction conditions were: 37 ° C water bath for 1 h.
  • the above method using the CRISPR / Cas9 system to target the knockout of the human TNFSF18 gene can be used for knockout of the human TNFSF18 gene.
  • the method for applying the CRISPR / Cas9 system to target the knockout of human TNFSF18 gene provides an experimental technology platform for in-depth exploration of the role of TNFSF18 gene, and can be used in the research and development of drugs related to abnormal expression of TNFSF18 gene.
  • FIG. 1 shows the T7 Endonuclease of Jurkat cells in the control and experimental groups. I test results chart.
  • a gRNA targeting TNFSF18 was designed, and its upstream sequence was 5’-ACCGCTGGTTAAGCCAATTGACAG -3 ', the downstream sequence is 5'-AAACCTGTCAATTGGCTTAACCAG-3'. After dissolving, 5 ⁇ l of each was mixed, heated at 95 ° C. for 5 minutes, and then naturally cooled to room temperature to form a double-stranded DNA with Bbs I sticky ends.
  • the px459 plasmid was digested with Bbs I, and the digested product was subjected to agarose gel electrophoresis. The gel was recovered and the concentration was determined.
  • the correct strain was sequenced and identified in Example 1, 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 endotoxin-free pxTNFSF18 plasmid.
  • Jurkat cells were seeded in a 35 mm petri dish with a seeding density of 50% and DMEM containing 10% fetal bovine serum (FBS) under 37 ° C and 5% CO2.
  • FBS fetal bovine serum
  • 2 ⁇ g of pxTNFSF18 plasmid was introduced according to the instructions of the Lipofectamine 2000 kit.
  • 1 ⁇ g / ml puromycin was added to screen for 7 d. After the screening was completed, the concentration of puromycin was reduced to 0.5 ⁇ g / ml and the cells were expanded.
  • Untreated Jurkat cells and Jurkat cells introduced with the pxTNFSF18 plasmid were inoculated into six-well plates. After the cells were full, genomic DNA was extracted, and then the high-fidelity PCR enzyme PrimeSTAR HS was used to amplify the expected sites of gene editing. Electrophoresis The PCR product was recovered.
  • T7 Endonuclease I was digested for 1 h at 37 ° C 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 strips. The band indicates that the gRNA sequence targeting the TNFSF18 gene guided the CRISPR / Cas9 system to successfully edit the human TNFSF18 gene.
  • the method for applying the CRISPR / Cas9 system to target the knockout of human TNFSF18 gene provides an experimental technology platform for in-depth exploration of the role of TNFSF18 gene, and can be used in the research and development of drugs related to abnormal expression of TNFSF18 gene.

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

Abstract

L'invention concerne un procédé d'invalidation ciblée d'un gène TNFSF18 humain par application d'un système CRISPR/Cas9.
PCT/CN2018/091726 2018-06-16 2018-06-16 Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9 Ceased WO2019237396A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091726 WO2019237396A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091726 WO2019237396A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9

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WO2019237396A1 true WO2019237396A1 (fr) 2019-12-19

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PCT/CN2018/091726 Ceased WO2019237396A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation ciblée d'un gène tnfsf18 humain par application d'un système crispr/cas9

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106987560A (zh) * 2017-03-29 2017-07-28 中国农业科学院上海兽医研究所 Rk‑13细胞hb基因敲除稳定株的构建方法
EP3239298A1 (fr) * 2014-12-26 2017-11-01 Riken Procédé d'invalidation génique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3239298A1 (fr) * 2014-12-26 2017-11-01 Riken Procédé d'invalidation génique
CN106987560A (zh) * 2017-03-29 2017-07-28 中国农业科学院上海兽医研究所 Rk‑13细胞hb基因敲除稳定株的构建方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHANG, LIXIAN ET AL.: "#IJfflCRISPR/Cas9n double (Knocking out of Human DNAH2 Gene in U20S Cells by CRISPR/ Cas9n Double Nick System", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 33, no. 2, 25 February 2017 (2017-02-25), pages 284 - 293, XP055671479, ISSN: 1000-3061, DOI: 10.13345/j.cjb.160273 *
JAFARI, N. ET AL.: "CRISPR-Cas9 Mediated NOX4 Knockout Inhibits Cell Proliferation and Invasion in HeLa Cells", PLOS ONE, vol. 12, no. 1, 18 January 2017 (2017-01-18), pages 1 - 13, XP055671480, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0170327 *

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