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CRISPR-Cas9 targeting knockout of human breast cancer cell RASSF2 gene and specific sgRNA of RASSF2 gene

A human breast cancer cell, specific technology, applied in the direction of DNA/RNA fragment, retroRNA virus, genetic engineering, etc., can solve the problems of loss of tumor suppressor function, decreased tumor suppressor function of RASSF2, decreased transcriptional activity of RASSF2, etc.

Inactive Publication Date: 2018-04-10
OBIO TECH SHANGHAI CORP LTD
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The loss of the tumor suppressor function of RASSF2 is mainly due to the methylation of the CpG island in the promoter region of the RASSF2 gene, which leads to the reduction or loss of the transcriptional activity of RASSF2
Ultimately leading to reduced tumor suppressor function of RASSF2

Method used

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  • CRISPR-Cas9 targeting knockout of human breast cancer cell RASSF2 gene and specific sgRNA of RASSF2 gene
  • CRISPR-Cas9 targeting knockout of human breast cancer cell RASSF2 gene and specific sgRNA of RASSF2 gene
  • CRISPR-Cas9 targeting knockout of human breast cancer cell RASSF2 gene and specific sgRNA of RASSF2 gene

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Embodiment 1

[0042] 1. Construction of knockout RASSF2 plasmid using CRISPR / Cas9 technology

[0043] 1.1 sgRNA oligonucleotide chain synthesis

[0044]Using the CRISPR online design tool (http: / / crispr.mit.edu / ) according to the scoring system, design a 20bp sgRNA on exon 2 of RASSF2, and verify that there is no non-specific gene by BLAST. Add CACC to the 5' end of the coding strand template, and add AAAC to the 3' end of the non-coding strand template, which is complementary to the cohesive end formed after digestion with BsmBI, and design a pair of CRISPR oligonucleotide chains, see Table 1, Table 1 RASSF2 targeting position Dot and sgRNA oligonucleotide sequence.

[0045]

[0046] 1.2 Vector construction

[0047] 1.2.1 Use BsmBI to digest 2 μg lentiCRISPRv2 plasmid (purchased from Addgene), 2h, 37°C, the enzyme digestion system is as follows:

[0048]

[0049] 1.2.2 Use the GENRY gel recovery kit to purify the digested plasmid product, and operate according to the instructions....

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Abstract

The invention discloses CRISPR-Cas9 targeting knockout of human breast cancer cell RASSF2 gene and specific sgRNA of RASSF2 gene. According to the method, the sgRNA of specific targeting RASSF2 gene is obtained, wherein the base sequence of the sgRNA is represented by SEQ ID NO.1; construction of the sgRNA of RASSF2 gene into a lentiviral vector system is carried out, wherein the lentiviral vectorsystem contains Cas9 protein; and at last, human breast cancer cell MDA-MB-231 is infected with the CRISPR-Cas9 lentivirus containing the sgRNA so as to obtain a cell strain with obviously reduced RASSF2 protein expression level. The operation and the steps are simple; the sgRNA targeting performance is excellent; cutting efficiency on RASSF2 gene is high; the constructed CRISPR-Cas9 lentiviral vector system is high in knockout efficiency, and is capable of realizing specific knockout of RASSF2 gene to obtain human breast cancer cells without RASSF2 gene, so that powerful instrument is provided for study on the action mechanisms of RASSF2 in breast cancer celles.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically, CRISPR-Cas9 targets knockout of human breast cancer cell RASSF2 gene and its specific sgRNA. Background technique [0002] RASSF2 (ras association domain family 2), a member of the RASSF family, is located at 20pl3 of the human genome, with a total length of about 43.6kb and a encoded protein of about 37.8kD. The protein expression is mainly enriched in the nucleus. In 1996, Nagase et al. found 40 unidentified cDNA clone fragments when studying the human coding region, and named these 40 transcripts KIAA0161200. During the research on one of them named KIAA0168, it was found that its coding sequence contained the unique RA domain of RASSF family members, so this gene was named RASSF2. [0003] In the human genome, the demethylation status of CpG islands in promoter regions is required for gene transcription. The loss of the tumor suppressor function of RASSF2 is mainly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/867
CPCC07K14/4703C12N9/22C12N15/113C12N15/86C12N15/907C12N2310/10C12N2740/15043C12N2800/107C12N2810/10
Inventor 马佩敏孙子豪杨兴林潘讴东
Owner OBIO TECH SHANGHAI CORP LTD
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