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WO2019222970A1 - Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant - Google Patents

Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant Download PDF

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Publication number
WO2019222970A1
WO2019222970A1 PCT/CN2018/088282 CN2018088282W WO2019222970A1 WO 2019222970 A1 WO2019222970 A1 WO 2019222970A1 CN 2018088282 W CN2018088282 W CN 2018088282W WO 2019222970 A1 WO2019222970 A1 WO 2019222970A1
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Prior art keywords
gene
human
grna
crispr
cells
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Ceased
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PCT/CN2018/088282
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English (en)
Chinese (zh)
Inventor
毛吉炎
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Biocan Technologies Co Ltd
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Shenzhen Biocan Technologies Co Ltd
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Priority to PCT/CN2018/088282 priority Critical patent/WO2019222970A1/fr
Publication of WO2019222970A1 publication Critical patent/WO2019222970A1/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
    • C12N15/11—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

Definitions

  • the invention belongs to the field of genetic engineering and biomedical technology, and particularly relates to a CRISPR / Cas9 targeted knockout human CD226 gene and a specific gRNA thereof.
  • CD226 molecule is a widely expressed immune system such as T cells, NK cells, NK T cells, activated vascular endothelial cells, megakaryocyte / platelet lineage, monocytes, thymocytes, certain B cell subsets, some hematopoietic stem cells and mast cells.
  • T cells T cells
  • NK cells NK T cells
  • activated vascular endothelial cells megakaryocyte / platelet lineage
  • monocytes thymocytes
  • thymocytes certain B cell subsets
  • some hematopoietic stem cells and mast cells include CD226 molecule.
  • CD226 and its ligands participates in the activation, differentiation and cytotoxicity of CTL and NK cells, regulates the proliferation and differentiation of CD4 + T cells, mediates the adhesion of endothelial cells and other cells, promotes the activation and aggregation of platelets, and participates in hematopoietic regulation Mediates the degranulation of mast cells, participates in the anti-apoptotic effects of NKT cells and thymocytes, the maturation of dendritic cells, the formation of immune synapses and synapses, and has important potential in immunotherapy of tumors.
  • Intensive translational research has been conducted, but the lack of means for targeted knock-out of CD226 gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a CRISPR / Cas9 targeted knockout of human CD226 gene and its specific gRNA.
  • the gRNA sequence can be used to knock out the human CD226 gene, thereby suppressing or eliminating the expression of CD226.
  • the present invention has the following advantages and effects:
  • the invention designs and synthesizes two single-stranded oligo sequences according to the gRNA guide sequence, anneals to form a double strand, and then connects with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will find a match under the guidance of the gRNA.
  • the DNA sequence was cut to achieve the CD226 gene knockout.
  • Figure 1 shows the Western Blot results of Jurkat cells in the control and experimental groups.
  • human CD226 gene was found in GenBank, and potential target sites were designed in the exon region of human CD226 gene.
  • online design tools and gRNA design principles we evaluated the high-scoring target sites on the human CD226 gene sequence to design the gRNA.
  • the sequence is shown in SEQ ID NO.1, and then the CACC was added to the 5 ′ end to obtain the positive oligo. Nucleotides, and add AAAC to the 5 'end of its reverse complement.
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, denatured at 95 ° C, and annealed to form a double-stranded DNA molecule that can be ligated into the px459 vector.
  • the px459 vector was treated with Bbs I enzyme at 37 ° C for 1 h, and then electrophoresed on 1% agarose to recover the digested product.
  • the double-stranded DNA molecule that can be ligated into the px459 vector obtained in Example 1 was ligated with the px459 vector using T4 DNA ligase.
  • the ligation system (10 ⁇ l) was: annealed double stranded (CD226-gRNA) 2 ⁇ l, px459 vector 2 ⁇ l, 10 ⁇ T4 DNA Ligase Buffer 1 ⁇ l, T4 DNA Ligase 1 ⁇ l, ddH2O made up to 10 ⁇ l; connection conditions: 16 ° C overnight.
  • the ligation product is transformed into competent cells Stbl3.
  • the specific transformation method is: take out the competent cells Stbl3 at -80 ° C, and dissolve them in an ice bath; then take 1 ⁇ l of the above-mentioned ligation products to 50 ⁇ l of competent cells and mix for 30 minutes on ice; Do not shake during 42 s water bath for 60 s; cool in ice bath for 2 min; then add 800 ⁇ l LB medium and shake at 37 °C for 30 min; LB plate coated with 100 ⁇ g / ml ampicillin and culture overnight. After picking positive clones Shake at 37 ° C overnight for expansion and send for sequencing. The correct sequencing was the Cas9 vector required to target the CD226 gene, and was named px459-CD226 vector.
  • 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-CD226 vector without endotoxin.
  • Jurkat cells were resuscitated. The cells were placed in a 10% FBS + DMEM culture flask and cultured in a 37 ° C, 5% CO2 incubator. One day before transfection, the recovered cells were subcultured.
  • Jurkat cells without any treatment were used as the control group, and the cells obtained in Example 4 were used as the experimental group.
  • 100-200 ⁇ l of 5 ⁇ SDS-PAGE loading buffer was added, and the mixture was boiled in boiling water for 5 minutes.
  • 15 ⁇ l of the loaded SDS- PAGE protein electrophoresis After electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skimmed milk powder for 2 h, place the blocked PVDF membrane in rabbit anti-human CD226 antibody, rinse the buffer 3 times, and then transfer the membrane to goat anti-rabbit secondary antibody Buffer, incubate at room temperature for 60 min, and rinse 4 times with rinsing buffer.
  • the present invention has the following advantages and effects:
  • the invention designs and synthesizes two single-stranded oligo sequences according to the gRNA guide sequence, anneals to form a double strand, and then connects with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will find a match under the guidance of the gRNA.
  • the DNA sequence was cut to achieve the CD226 gene knockout.

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

Abstract

L'invention concerne un ARNg spécifique pour l'inactivation ciblée d'un gène CD226 humain, deux sites cibles étant conçus sur le gène CD226 humain selon le principe de conception de l'ARNg CRISPR/Cas9, et un nucléotide oligomère correspondant étant synthétisé puis construit sur un vecteur px459. Le gène CD226 humain peut être efficacement inactivé à l'aide d'un système CRISPR/Cas9 dirigé par un vecteur recombinant dans des cellules Jurkat. Le système CRISPR/Cas9 dirigé par ARNg fourni est censé trouver des applications dans de nouveaux médicaments destinés à traiter des tumeurs.
PCT/CN2018/088282 2018-05-24 2018-05-24 Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant Ceased WO2019222970A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088282 WO2019222970A1 (fr) 2018-05-24 2018-05-24 Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/088282 WO2019222970A1 (fr) 2018-05-24 2018-05-24 Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant

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WO2019222970A1 true WO2019222970A1 (fr) 2019-11-28

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PCT/CN2018/088282 Ceased WO2019222970A1 (fr) 2018-05-24 2018-05-24 Inactivation ciblée de crispr/cas9 du gène cd226 humain et arng spécifique correspondant

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104651392A (zh) * 2015-01-06 2015-05-27 华南农业大学 一种利用CRISPR/Cas9系统定点突变P/TMS12-1获得温敏不育系的方法
CN105637087A (zh) * 2013-09-18 2016-06-01 科马布有限公司 方法、细胞与生物体
CN105647969A (zh) * 2016-02-16 2016-06-08 湖南师范大学 一种基因敲除选育stat1a基因缺失型斑马鱼的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105637087A (zh) * 2013-09-18 2016-06-01 科马布有限公司 方法、细胞与生物体
CN104651392A (zh) * 2015-01-06 2015-05-27 华南农业大学 一种利用CRISPR/Cas9系统定点突变P/TMS12-1获得温敏不育系的方法
CN105647969A (zh) * 2016-02-16 2016-06-08 湖南师范大学 一种基因敲除选育stat1a基因缺失型斑马鱼的方法

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