Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof

A key region, specific technology, applied in the field of gRNA

Inactive Publication Date: 2017-02-22
ZUNYI MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The key region of the human ezrin gene enhancer is a non-transcribed region, which cannot be studied by traditional RNA interference methods

Method used

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  • Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof
  • Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof
  • Method for CRISPR/Cas9 targeted knockout of human ezrin gene enhancer key region and specific gRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 gRNA design and vector construction targeting the key region of human ezrin gene enhancer

[0026] 1. Design of gRNA targeting the key region of enhancer of human ezrin gene and synthesis of oligonucleotide chain

[0027] Find the human ezrin gene sequence from Genebank ( http: / / www.ncbi.nlm.nih.gov / gene / 7430 ), using the online software http: / / www.e-crisp.org / E-CRISP / The gRNA target sites were designed to be located upstream and downstream of the enhancer key region (–1297 / –1186) of the human ezrin gene, respectively. In the present invention, the DNA sequence corresponding to the gRNA is also called the gRNA sequence, which is the target site of the gRNA on the target gene. The oligonucleotide chain (Oligo DNA) corresponding to the gRNA is in accordance with the 5'-G(N) 20The PAM structure (protospacer adjacent motif) of NGG-3' is the design principle, and the sequence with a higher score is selected. If the first base at the 5' end of the forward ol...

Embodiment 2

[0036] Example 2 Cell culture and CRISPR / Cas9 recombinant plasmid transfection

[0037] Human esophageal cancer EC109 cells were grown adherently in DMEM medium containing 10% inactivated fetal bovine serum, digested with digestive solution containing 0.25% trypsin and 0.02% EDTA, and subcultured. Cells were seeded in 96-well cell culture plates, 100 μl per well. After 24 hours, the cells can be used for transfection when the confluence rate reaches 50%-60%. Co-transfection of plasmids pX459-sgRNA1 and pX459-sgRNA2 by liposome method, transfection steps refer to Lipofectamine TM 2000 Transfection Reagent instructions were performed.

Embodiment 3

[0038] Example 3 Targeted knockout detection of key regions of ezrin enhancer in human esophageal cancer cells

[0039] The recombinant plasmids pX459-sgRNA1 and pX459-sgRNA2 were co-transfected into esophageal cancer EC109 cells for 48 hours, and the cells were collected directly without resistance selection. Using cell genome DNA as a template, PCR amplifies the DNA sequence of the ezrin gene enhancer. The PCR primers are located upstream and downstream of the sequence to be knocked out, and the primer sequences are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0040] The sequences of gRNA1 and gRNA2 are respectively located on both sides of the key region of the ezrin enhancer. It is estimated that the amplified fragment of non-mutated genomic DNA is 766 bp (ezrin gene -1543 / -778 sequence); the mutant genomic DNA is deleted by 147 bp (ezrin gene -1319 / -1173 sequence ), the amplified fragment is about 619bp in length. The results of agarose gel electrophoresis of PCR products ar...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to a method for CRISPR/Cas9 targeted knockout of a human ezrin gene enhancer key region and specific gRNA thereof. In the method, according to the design principle of CRISPR/Cas9, two target sites are designed at the upstream and the downstream of the human ezrin gene enhancer key region, corresponding oligonucleotide sequences are synthesized and then connected to a carrier pX459 to construct recombinant plasmids, the recombinant plasmids are transfected with a human esophagus cancer cell line, and then specific knockout of the human ezrin gene enhancer key region can be achieved. The method and the specific gRNA have great significance for study on clinical tumor therapy with the human ezrin gene enhancer as the target.

Description

technical field [0001] The present invention belongs to the field of molecular biology, and specifically relates to a method for CRISPR / Cas9 targeted deletion of a key region of the ezrin gene enhancer of human esophageal cancer cells and a gRNA used for targeting the key region of the enhancer of the human ezrin gene. Background technique [0002] CRISPR / Cas (clustered regularly interspaced short palindromic repeats-associated) is the natural immune system of many bacteria and most archaea. It specifically recognizes invading viruses and nucleic acids, and uses Cas protein to cut them, so as to achieve self-protection. immunity. The CRISPR / Cas9 system draws on the defense strategy of bacteria. It uses gRNA (guide RNA) to find a specific DNA sequence, and then uses Cas9 endonuclease to cut the target DNA, causing double-strand breaks. In the absence of a template, non-isotropic Source end-joining, resulting in DNA deletion mutations (Shalem O, Sanjana NE, Hartenian E, et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85
CPCC12N15/113C07K14/47C12N15/85C12N2310/10
Inventor 高书颖张青峰郭晓龙
Owner ZUNYI MEDICAL UNIVERSITY
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