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Gene knockout test kit and method for rapidly screening sgRNA

A gene knockout and kit technology, which is applied in the direction of retroRNA virus, genetic engineering, plant gene improvement, etc., can solve the problem of low activity of sgRNA-mediated genome modification, and achieve the effect of shortening the experimental cycle and cost, and simple operation

Inactive Publication Date: 2017-04-26
SHANGHAI REPODX BIOTECH CO LTD
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are few methods for measuring the activity of sgRNA-mediated genome modification. Most laboratories mainly optimize the design of sgRNA, and the determination of sgRNA activity mainly needs to be verified by experiments, not just theoretical design optimization.

Method used

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  • Gene knockout test kit and method for rapidly screening sgRNA
  • Gene knockout test kit and method for rapidly screening sgRNA
  • Gene knockout test kit and method for rapidly screening sgRNA

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Embodiment 1

[0064] 1. Construction of pCAG-target-mCherry-Luc plasmid vector

[0065] The construction of the target gene pCAG-target-mCherry-Luc fluorescence-luciferase double reporter gene plasmid of sgRNA screening system in the present invention, wherein reagent A contains pCAG-mCherry-Luc fluorescence-luciferase double reporter gene plasmid, adopts 人工合成人源RPA3靶基因DNA CGCGGATCCGGTGCCGAGAAATCTGCCCCATAGACACCCGCGGGGCGCACAGTTTCAGTCGTCCGTGGGTTTCCCGCCAGCCGCAGTCTTGGACCATAATCATGGTGGACATGATGGACTTGCCCAGGTCGCGCATCAACGCCGGCATGCTAGCTCAATTCATCGACAAGCCTGTCTGCTTCGTAGGGAGGCTGGAAAAGATTCATCCCACCGGAAAAATGTTTATTCTTTCAGATGGAGAAGGAAAAAATGGAACCATCGAGTTGATGGAACCCCTTGATGAAGAAATCTCTGGAATTGTGGAAGTGGTTGGAAGAGTAACCGCCAAGGCCACCATCTTGTGTACATCTTATGTCCAGTTTAAAGAAGATAGCCATCCTTTTGATCTTGGACTTTACAATGAAGCTGTGAAAATTATCCATGACTTCCGAATTCCGG(SEQ ID NO:2),靶基因DNA的两端含有BamHI及EcoRI酶切位点,其中5端为BamHI,3端为EcoRI;pCAG-mCherry-Luc质粒和 The artificially synthesized target gene DNA was double-digested with BamHI and EcoRI, mixed, ligated with T4 DNA ...

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Abstract

The invention discloses a gene knockout test kit and method for rapidly screening sgRNA. The test kit includes a kit body, a reagent A and a reagent B. The test kit utilizes pCAG-mCherry-Luc plasmids obtained through fusion of full-length sequences-containing firefly luciferase and green fluorescent protein C terminals, targeted genomic sequences are inserted in the fronts of green fluorescent protein initiation codons, finally the plasmids and expression plasmids of sgRNA and CRISPR-Cas9 transfect HEK293FT cells together, through red fluorescence protein brightness observation and luciferase viability measurement, qualitative or quantitative measurement of sgRNA mediated genome modification activity is quickly conducted, and through a conventional method in the field, the effectiveness of an sgRNA screening system in a slow virus system is verified. The test kit and the method have the advantages that an analog genome of any target gene can be constructed simply and rapidly, and the sgRNA activity is evaluated more quickly and simply and accurately quantified.

Description

technical field [0001] The invention relates to the field of genome modification activity detection, in particular to a gene knockout kit and method for rapidly screening sgRNA. Background technique [0002] With the development and application of whole genome sequencing technology, more and more genome sequences have been published. But obtaining the genome sequence is not the ultimate goal of people, and interpreting the function of genes is the significance of studying these genes. The most direct and reliable way to study gene function is to carry out targeted modification of the genome. [0003] The CRISPR-Cas9 system is a new genome-directed editing technology that emerged after the ZFNs and TALENs technologies. Different from ZFNs and TALENs technologies, the recognition of target sites by CRISPR-Cas9 technology relies on complementary base pairing between nucleic acids, and any 20bp target site sequence immediately following PAM (NGG) can be edited, and its target ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/867
CPCC12N15/85C12N15/86C12N2740/15043C12N2800/107C12N2810/10
Inventor 卿志荣郑帮郭帅修朝阳
Owner SHANGHAI REPODX BIOTECH CO LTD
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