SgRNA screening for DJ-1 gene editing as well as vector and application of sgRNA

A technology of DJ-1 and genes, applied in fields including sgRNA and its vectors and applications, can solve problems such as time-consuming, laborious, inaccurate, and high requirements for equipment and equipment

Active Publication Date: 2018-10-09
ZHANGJIAGANG INST OF IND TECH SOOCHOW UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To evaluate the specificity and effectiveness of sgRNA for a specific target sequence, the following methods are currently widely used, such as T7 endonuclease I experiment, PCR product sequencing, Digenome-seq technology, GUIDE-seq technology, HTGTS technology, IDLV method and Prediction by online tools, but these methods are usually time-consuming and labor-intensive, requiring high equipment requirements or inaccurate predictions

Method used

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  • SgRNA screening for DJ-1 gene editing as well as vector and application of sgRNA
  • SgRNA screening for DJ-1 gene editing as well as vector and application of sgRNA
  • SgRNA screening for DJ-1 gene editing as well as vector and application of sgRNA

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Experimental program
Comparison scheme
Effect test

Embodiment

[0058] For the sgRNA of the DJ-1 gene, the sequence of the sgRNA for the DJ-1 gene is SEQ ID NO. 1, specifically 5'-ACATCACGGCTACACTGTACTGG-3', referred to as sgRNA4.

Embodiment 2

[0137] The pSpCas9(BB)-2A-GFP plasmid loaded with the sgRNA (SEQ ID NO. 1) of the present invention was constructed according to the above-mentioned conventional method. According to the above conventional transfection system, pipette 0.8μg of successfully constructed pSpCas9(BB)-2A-GFP plasmid and 2μL of Lipo2000 into centrifuge tubes with 50μL of preheated OMEM medium, mix gently, and let stand for 5min; Then mix the two tubes of solution, shake slightly to mix, and let stand for 20 min. Aspirate the cell culture medium, wash with PBS solution, add 100μL of mixed solution and 100μL of preheated OMEM medium to each well of the 24-well plate, and put in 5% CO 2 Incubate at 37℃ for 4h, then add 200μL of complete medium, incubate for 12h, suck off the liquid in the culture plate, add 1mL of complete medium, and put in 5% CO 2 Incubate at 37°C for 48h. At the same time, the experimental group (containing the sgRNA of SEQ ID NO.1) and the negative control group (without sgRNA) were...

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PUM

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Abstract

The invention discloses an sgRNA for a DJ-1 gene. A prokaryotic evaluation system such as a white-to-blue clone formation assay confirms the editing efficiency of the sequence, and proves that the sequence provided by the invention is excellent in editing efficiency and provides a reference for a future DJ-1 gene therapy; cell proliferation assays show that cell proliferation of cell lines slows down after the DJ-1 gene is knocked out; and at the same time, an effective cell tool is provided for a further DJ-1 mechanism research.

Description

Technical field [0001] The present invention belongs to gene technology, and specifically relates to sgRNA directed to DJ-1 gene, has high editing efficiency, and contains sgRNA and its carrier and application. Background technique [0002] DJ-1 (PARK7) is a more common autosomal recessive inherited Parkinson's disease gene, and its pathogenic mechanism for Parkinson's disease is not clear and needs to be studied. The CRISPR / Cas9 (Clustered Regularly Interspaced ShortPalindromic Repeats / Cas9) gene editing system is the third-generation gene editing system developed from ZFNs and TALENs. It is discovered from the adaptive immune defense system of bacteria and is used to combat foreign DNA and The invading virus has been widely used in the field of biomedicine. The characteristics of CRISPR / Cas9 are its simple design, low cost, recognition not affected by gene methylation, high editing efficiency, simultaneous targeting of multiple targets and arbitrary sequences containing PAM si...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/63A61K31/7105A61P25/16
CPCA61K31/7105A61P25/16C12N15/113C12N15/63C12N2310/10C12N2310/20
Inventor 孙万平奚邦生刘婉婉陶静马建婷
Owner ZHANGJIAGANG INST OF IND TECH SOOCHOW UNIV
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