WO2020000457A1 - Séquence de ciblage d'arng qui cible spécifiquement le gène kat13d humain et son utilisation - Google Patents

Séquence de ciblage d'arng qui cible spécifiquement le gène kat13d humain et son utilisation Download PDF

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Publication number
WO2020000457A1
WO2020000457A1 PCT/CN2018/093867 CN2018093867W WO2020000457A1 WO 2020000457 A1 WO2020000457 A1 WO 2020000457A1 CN 2018093867 W CN2018093867 W CN 2018093867W WO 2020000457 A1 WO2020000457 A1 WO 2020000457A1
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WIPO (PCT)
Prior art keywords
kat13d
gene
grna
human
vector
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PCT/CN2018/093867
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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/CN2018/093867 priority Critical patent/WO2020000457A1/fr
Publication of WO2020000457A1 publication Critical patent/WO2020000457A1/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

Definitions

  • the invention belongs to the technical field of gene editing, and particularly relates to a gRNA guide sequence that specifically targets a human KAT13D gene and an application thereof.
  • Circadian rhythm is a kind of biological rhythm. It is a rhythm that appears under the combined effect of exogenous and endogenous factors. Its endogenous factor is a closed-loop oscillatory system composed of a set of biorhythmic genes and their gene products.
  • Biorhythm genes include KAT13D, Per1, Per2, Per3, Cry1, Cry2, BMAL1, Timless, etc., which play a key role in maintaining the body's body temperature, breathing, sleep, eating, blood pressure, blood glucose, endocrine and many other homeostasis.
  • KAT13D gene plays a central role in this system. Therefore, the study of the CLOCK gene is very important, but the lack of means for targeted knock-out of KAT13D gene expression in the prior art has caused certain obstacles to the progress of related research.
  • the present invention provides a gRNA targeting sequence that specifically targets the human KAT13D gene.
  • the gRNA targeting sequence can be used to knock out the human KAT13D gene, thereby suppressing or eliminating the expression of KAT13D.
  • Yet another object of the present invention is to provide an application of the gRNA-directed sequence specifically targeting the human KAT13D gene.
  • a gRNA targeting sequence that specifically targets the human KAT13D gene is KAT13D-gRNA, and its nucleotide sequence is:
  • KAT13D-gRNA 5’- CAAACGCCAGCGGCGGTGAC -3 ’;
  • a method for knocking out the human KAT13D gene using the CRISPR / Cas9 system includes the following steps:
  • step b ligating the double strand prepared in step a with the Cas9 vector to obtain a recombinant knockout expression vector
  • the recombinant knockout expression vector prepared in step b is transfected into the target cells, and puromycin is selected to obtain cells that have successfully knocked out the KAT13D gene.
  • the Cas9 vector in step b is a px459 vector
  • the target cell in step c is a C6 cell
  • the concentration of puromycin in step c is 1.0 ⁇ g / ml.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will be found under the guidance of the gRNA
  • the matching DNA sequence is cut to realize the KAT13D gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.
  • Figure 1 shows the Western Blot results of C6 cells in the control and experimental groups.
  • Example 1 Targeting KAT13D genetic gRNA design
  • a gRNA targeting human KAT13D gene was designed.
  • the 20nt oligonucleotide gRNA targeting sequence is: KAT13D-gRNA: 5'- CAAACGCCAGCGGCGGTGAC -3 ', then add CACC to its 5' end to obtain a forward oligonucleotide, and 5 'to its reverse complementary sequence Add AAAC to the end.
  • 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 has a Bbs I digestion site, which is digested with Bbs I.
  • the digestion system (20 ⁇ l) is: Bbs I 1 ⁇ L; 10 ⁇ FastDigest buffer 2 ⁇ L; plasmid 1 ⁇ g; ddH2O supplemented to 20 ⁇ l; digestion conditions are: : Digestion for 1 h at 37 ° C. After the digestion is completed, the gel is recovered and purified.
  • the digested vector px459 and the annealed double strand obtained in Example 1 were ligated with T4 ligase.
  • the ligation system (10 ⁇ l) was: annealed double strand (KAT13D-gRNA) 2 ⁇ l, px459 vector 2 ⁇ l, 10 ⁇ T4 DNA Ligase Buffer 1 ⁇ l, T4 DNA Ligase 1 ⁇ l, ddH2O to make up to 10 ⁇ l; ligation conditions: ligation at 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 is the required Cas9 vector targeting KAT13D gene, named px459-KAT13D 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-KAT13D vector without endotoxin.
  • Embodiment 4 C6 Transfection of cells
  • C6 cells should be cultured until the fusion rate of C6 cells reaches 50% to 60%.
  • the optimal transfection time is 12 to 18 hours after inoculation. Change the fresh culture medium before transfection. Add 3 ml medium to a 60 mm culture dish. Transfection 4 ⁇ g of px459-KAT13D plasmid was introduced according to the instructions of the Lipofectamine 2000 kit. 48 hours after transfection, 1 ⁇ g / ml puromycin was added for screening for 7 days. After the screening was completed, the concentration of puromycin was reduced to 0.5 ⁇ g / ml and the cells were expanded.
  • Embodiment 5 Western Blot Detection of transfection effect
  • C6 cells without any treatment were used as the control group, and the cells selected 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 SDS was loaded.
  • -PAGE protein electrophoresis After electrophoresis, semi-dry transfer with conventional protein, blocking with 10% skim milk powder for 2 h, put the blocked PVDF membrane in rabbit anti-human KAT13D 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 Western blot membrane was developed and detected by ECL, and the results are shown in FIG. 1. It can be seen that the KAT13D protein band cannot be detected by Western Blot in the KAT13D frameshift mutant C6 cells, while the control group has a KAT13D protein band, indicating that the gRNA sequence used for knocking out the KAT13D gene of human cells can achieve KAT13D Gene knockout.
  • the present invention has the following advantages and effects:
  • the present invention designs and synthesizes two single-stranded oligo sequences according to the gRNA-directed sequence, anneals to form a double-strand, and then ligates with the Cas9 vector.
  • the Cas9 vector is used to introduce the gRNA and CRISPR system into the target cell.
  • the Cas9 protein will be found under the guidance of the gRNA.
  • the matching DNA sequence is cut to realize the KAT13D gene knockout.
  • the vector contains a puromycin resistance gene. Puromycin can be used to screen cells, and cells that have not been transferred into the vector can be screened out.

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

Abstract

L'invention concerne une séquence de ciblage d'ARNg qui cible spécifiquement le gène KAT13D humain et son utilisation, la séquence nucléotidique de la séquence de ciblage d'ARNg étant 5'-CAAACGCCAGCGGCGGTGAC-3'. L'inactivation du gène KAT13D est obtenue par introduction de l'ARNg et du système CRISPR dans des cellules cibles à l'aide du vecteur Cas9.
PCT/CN2018/093867 2018-06-29 2018-06-29 Séquence de ciblage d'arng qui cible spécifiquement le gène kat13d humain et son utilisation WO2020000457A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2018/093867 WO2020000457A1 (fr) 2018-06-29 2018-06-29 Séquence de ciblage d'arng qui cible spécifiquement le gène kat13d humain et son utilisation

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Application Number Priority Date Filing Date Title
PCT/CN2018/093867 WO2020000457A1 (fr) 2018-06-29 2018-06-29 Séquence de ciblage d'arng qui cible spécifiquement le gène kat13d humain et son utilisation

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WO2020000457A1 true WO2020000457A1 (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
CN101418293A (zh) * 2008-12-03 2009-04-29 暨南大学 微小rna-21的反义寡聚核苷酸及其应用
CN105979954A (zh) * 2013-11-13 2016-09-28 在配料公司 昼夜节律蛋白相关状况的治疗或预防
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

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101418293A (zh) * 2008-12-03 2009-04-29 暨南大学 微小rna-21的反义寡聚核苷酸及其应用
CN105979954A (zh) * 2013-11-13 2016-09-28 在配料公司 昼夜节律蛋白相关状况的治疗或预防
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

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