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WO2019237399A1 - Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé - Google Patents

Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé Download PDF

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Publication number
WO2019237399A1
WO2019237399A1 PCT/CN2018/091732 CN2018091732W WO2019237399A1 WO 2019237399 A1 WO2019237399 A1 WO 2019237399A1 CN 2018091732 W CN2018091732 W CN 2018091732W WO 2019237399 A1 WO2019237399 A1 WO 2019237399A1
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WO
WIPO (PCT)
Prior art keywords
c2orf40
gene
sgrna
crispr
cas9
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/091732
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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
Original Assignee
Shenzhen Biocan Technologies Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Biocan Technologies Co Ltd filed Critical Shenzhen Biocan Technologies Co Ltd
Priority to PCT/CN2018/091732 priority Critical patent/WO2019237399A1/fr
Publication of WO2019237399A1 publication Critical patent/WO2019237399A1/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 belongs to the technical field of gene editing, and particularly relates to a method for specifically knocking out the human C2orf40 gene by CRISPR-Cas9 and a specific sgRNA thereof.
  • C2orf40 was originally identified and cloned by Bi et al. They found that expression of C2orf40 in esophageal squamous cell carcinoma tissues and cell lines was significantly reduced compared to normal adult esophageal epithelium. Therefore, they believe that C2orf40 may be a new candidate tumor suppressor gene. In recent years, there have been more studies on C2orf40 in esophageal cancer, colon cancer, glioma and prostate cancer.
  • C2orf40 is a candidate tumor suppressor gene for esophageal squamous cell carcinoma (ESCC). It is believed that promoter hypermethylation may be the main mechanism of C2orf40 transcriptional inactivation in ESCC. It is believed that C2orf40 may be involved in regulating the expression of COX-2 by participating in the NF- ⁇ B pathway. Taking advantage of its anti-cancer function, it can be used as a potential therapeutic drug for ESCC. It has great potential for transformation and requires a lot of research. However, the lack of a plasmid that enhances the expression of the C2orf40 gene in the prior art has caused certain obstacles to the progress of related research.
  • ESCC esophageal squamous cell carcinoma
  • the purpose of the present invention is to provide a method for specifically knocking out the human C2orf40 gene and a specific sgRNA of the CRISPR-Cas9 with simple structure, reasonable design, and convenient use in response to the defects and deficiencies of the prior art.
  • Knockout can achieve a permanent effect; it provides highly efficient sgRNA; sgRNA only needs to synthesize a small amount of polynucleotide fragments, which can be produced in large quantities.
  • the CRISPR-Cas9 method for specifically knocking out the human C2orf40 gene and the specific sgRNA thereof according to the present invention adopt the following technical scheme:
  • the selected sgRNA add CCGG to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain the complementary strand of its corresponding DNA, and add AAAC to its 5' end to obtain a reverse oligonucleotide .
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse sequence oligonucleotide sequences of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, the PX330 vector could be ligated. Double-stranded annealing product.
  • the annealed sgRNA oligonucleotide double strand was ligated with the linearized PX330 plasmid to obtain the PX330-C2orf40 plasmid.
  • the invention also provides an sgRNA that specifically targets the C2orf40 gene, the sequence of which is as follows: ID NO. 1.
  • the present invention has the following advantages and effects:
  • the function of the antibody is only a temporary blocking effect.
  • the direct knockout of the C2orf40 gene in the present invention can achieve a permanent effect;
  • the present invention can be used to knock out multiple coding sequences of C2orf40;
  • Antibodies can only target extracellular targets, and the present invention can target both extracellular and intracellular targets;
  • Figure 1 is the structure of the PX330 carrier
  • Figure 2 shows the specific cleavage of human sgRNA / Cas9-mediated gene C2orf40 by T7EN1 digestion.
  • 1- untreated control group 293T cells 2- experimental group transfected with PX330-C2orf40 vector 293T cells.
  • the selected sgRNA sequence (SEQ ID No. 1), add CACC to its 5 'end to obtain a forward oligonucleotide; according to the selected sgRNA, obtain its complementary strand, and add AAAC to its 5' end to obtain a reverse oligonucleotide.
  • the above-mentioned forward oligonucleotide and reverse oligonucleotide were synthesized separately, and the forward and reverse oligonucleotides of the synthesized sgRNA oligonucleotide were denatured and annealed in pairs. After annealing, a double pair that can be ligated into the PX330 vector was formed. Stranded sgRNA oligonucleotide.
  • the denaturing and annealing system is:
  • the enzyme digestion system and conditions are as follows:
  • the double-stranded sgRNA oligonucleotides that can be ligated into the PX330 vector after denaturation and annealing were ligated with the linearized PX330 plasmid to obtain the PX330-C2orf40 plasmid.
  • connection system is as follows:
  • the high-fidelity PCR enzyme was amplified, and the PCR cleanup was purified to obtain the PCR recovered product.
  • 100 ng was uniformly diluted to 20 ⁇ L for denaturation and annealing. The procedures were: 95 ° C, 3min; 95-85 ° C, cooling at 2 ° C / s; 85-25 ° C, cooling at 0.1 ° C / s; 4 ° C hold.
  • the present invention has the following advantages and effects:
  • the function of the antibody is only a temporary blocking effect.
  • the direct knockout of the C2orf40 gene in the present invention can achieve a permanent effect;
  • the present invention can be used to knock out multiple coding sequences of C2orf40;
  • Antibodies can only target extracellular targets, and the present invention can target both extracellular and intracellular targets;

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

Abstract

L'invention concerne un procédé d'invalidation spécifique du gène C2orf40 humain par CRISPR-Cas9 et un ARNsg spécifique associé.
PCT/CN2018/091732 2018-06-16 2018-06-16 Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé Ceased WO2019237399A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091732 WO2019237399A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/091732 WO2019237399A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé

Publications (1)

Publication Number Publication Date
WO2019237399A1 true WO2019237399A1 (fr) 2019-12-19

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PCT/CN2018/091732 Ceased WO2019237399A1 (fr) 2018-06-16 2018-06-16 Procédé d'invalidation spécifique du gène c2orf40 humain par crispr-cas9 et arnsg spécifique associé

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014204724A1 (fr) * 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, modification et optimisation de systèmes guides tandems, méthodes et compositions pour la manipulation de séquence
CN104762321A (zh) * 2015-04-22 2015-07-08 东北林业大学 基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014204724A1 (fr) * 2013-06-17 2014-12-24 The Broad Institute Inc. Administration, modification et optimisation de systèmes guides tandems, méthodes et compositions pour la manipulation de séquence
CN104762321A (zh) * 2015-04-22 2015-07-08 东北林业大学 基于CRISPR/Cas9系统靶向敲除KHV基因的敲除载体构建方法及其crRNA原件

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, GUANGLEI: "Site-specific Editing of Pig Genome Using CRISPR/Cas9 and Dysregulation of DNA Methylation in Cloned Piglets", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE , AGRICULTURE SCIENCE AND TECHNOLOGY, 15 February 2017 (2017-02-15), pages 33 - 34, XP055672692, ISSN: 1674-022X *

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