Kit for knocking out glioblastoma DHC2 gene

A glioblastoma and kit technology, applied in the field of alleles, can solve the problems of low editing efficiency, low off-target effect, low knockout efficiency, large workload, etc., and achieve high gene editing efficiency, strong specificity, and off-target rate. low effect

Active Publication Date: 2018-03-27
NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, if the DHC2 gene of GBM cells is operated according to the conventional steps, the workload is heavy and the knockout efficiency is extremely low. It is necessary to develop improved gene editing methods and kits with high editing efficiency and low off-target effects.

Method used

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  • Kit for knocking out glioblastoma DHC2 gene
  • Kit for knocking out glioblastoma DHC2 gene
  • Kit for knocking out glioblastoma DHC2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of the kit containing knockout plasmid according to the present invention

[0037] 1. Target site oligo DNA (oligo DNA) synthesis, and add adapters.

[0038] Using the online tool website (http: / / crispr.mit.edu / ), select the CDS region common to all transcripts of the DHC2 gene (gene ID: 79659), and find the third exon where the CDS region is located for the target site Design multiple gRNA sequences for knockout experiments.

[0039] Finally, 3 gRNA sequences were determined, the corresponding oligo DNA sequences were synthesized according to the 3 gRNA sequences, and additional bases were added to the head of the target sequence, CACC was added to the forward sequence, and AAAC (the first base of the target sequence) was added to the reverse sequence. The base must be G. If the first base of the selected target sequence is not G, you can add a G before the target sequence. The oligo DNA sequence corresponding to each gRNA is as follows:

[00...

Embodiment 2

[0084] Example 2: Using the kit of the present invention to stably knock out the DHC2 gene of the GBM cell line U87 cells

[0085] 1. To explore the conditions of electroporation of U87 cells

[0086] U87 cells were purchased from the ATCC cell bank in the United States. After the U87 cells were made into a uniform single cell suspension, a part of the cells was mixed and counted by trypan blue staining (the viable cell rate was >95% for electroporation), and the cell suspension was calculated. Concentration; take 3.0×10 6 The cells were suspended in a sterile tube and centrifuged at 1000 rpm for 4 min; the cell pellet was taken, a certain amount of DPBS was added to it, and the pEGFP-N1 plasmid (total 30ug, de-endotoxin treatment) was added after mixing, so that the final total system was at About 330ul; after mixing, add it into 3 electric shock cups with the electric rotary gun head respectively, so that the liquid level of the nozzle is raised, and no bubbles are formed i...

Embodiment 3

[0138] Example 3: Screening of knockout plasmids

[0139] This kit contains a unique knock-out plasmid, which has been verified by the inventor for many times. Compared with other knock-out plasmids, the knock-out plasmid contained in this kit has the characteristics of low off-target rate and strong specificity; The inventors have repeatedly verified that according to the method provided by the kit, the DHC2 two alleles can be stably knocked out, and the gene editing efficiency is extremely high.

[0140] The following table lists other gRNA sequences designed and tested by the inventor and the editing of the DHC2 gene, but the experimental results are not satisfactory.

[0141] Table 1. Other gRNA sequences and editing of DHC2 gene

[0142]

[0143] To sum up, the inventors unexpectedly found knockout plasmids with surprising effects and their combinations in experiments, and thus propose the kit for knocking out DHC2 gene in glioblastoma according to the present inventi...

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Abstract

The invention relates to a kit for knocking out a glioblastoma DHC2 gene, and application of the kit, and a method for knocking out a glioblastoma DHC2 allele. The kit for knocking out the glioblastoma DHC2 gene comprises a first knockout plasmid and a second knockout plasmid, or a knockout plasmid and a third knockout plasmid, or comprises a first knockout plasmid, a second knockout plasmid and athird knockout plasmid, wherein a first guide RNA shown in a sequence SEQ ID NO: 1 is transcribed from the first knockout plasmid, and a second guide RNA shown in a sequence SEQ ID NO: 2 is transcribed from the second knockout plasmid; a third guide RNA shown in a sequence SEQ ID NO: 3 is transcribed from the third knockout plasmid. The kit for stably knocking out the DHC2 gene is firstly provided. The knockout plasmid included in the kit has the characteristics of low off-target rate and strong specificity. The kit can stably knock out two DHC2 alleles, and has extremely high gene editing efficiency.

Description

technical field [0001] The invention relates to a kit for knocking out DHC2 gene of glioblastoma cells, the application of the kit, and a method for knocking out DHC2 allele of glioblastoma cells. Background technique [0002] Glioma is the most common primary malignant brain tumor, and among all histological types of gliomas, glioblastoma (GBM) is the most common, the most malignant, and the worst in prognosis. Progression survival was only 6.9 months, and median overall survival was only 14.6 months. Although glioma research has made great progress at the level of gene analysis, molecular targeted drugs, immunotherapy, etc., have not yet made significant progress in GBM treatment. At present, the international treatment of glioma still mainly adopts total surgical resection with the maximum safety margin, supplemented by standardized concurrent chemoradiotherapy after surgery. Temozolomide (TMZ) is the preferred chemotherapy drug. However, GBM will develop treatment resi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/113C12N5/10C12N15/90
CPCC07K14/47C12N15/113C12N15/63C12N15/907C12N2310/10
Inventor 刘亚伟姚夙
Owner NANFANG HOSPITAL OF SOUTHERN MEDICAL UNIV
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