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Targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats)/Cas9 slow virus system and application thereof

A technology of lentivirus and lentiviral vector, which is applied in the direction of retroRNA virus, DNA/RNA fragments, applications, etc., and can solve the problems of incomplete knockout, low efficiency, and unsuitability for long-term inhibition research

Inactive Publication Date: 2016-10-26
THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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Problems solved by technology

However, RNA interference technology is still used in the study of bladder cancer-related genes, and its main disadvantage is that interference at the RNA level can only be knocked down, not completely knocked out, and the efficiency is low, so it is not suitable for long-term inhibition research

Method used

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  • Targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats)/Cas9 slow virus system and application thereof
  • Targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats)/Cas9 slow virus system and application thereof
  • Targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats)/Cas9 slow virus system and application thereof

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Experimental program
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Effect test

Embodiment 1

[0051] 1. Construction of knockout FTO plasmid using CRISPR / Cas9 technology

[0052] 1.1 sgRNA oligonucleotide chain synthesis

[0053] Use the CRISPR online design tool (http: / / crispr.mit.edu / ) to design three 20bp sgRNAs (sp1, sp2 and sp3) on exon 3 of FTO according to the scoring system, and verify that there is no non-specificity by BLAST sex gene. The core sequences on the exons were found according to these two criteria: FTOsgRNAsp1, FTOsgRNAsp2 and FTOsgRNAsp3. CACC was added to the 5' end of the coding strand template, and AAAC was added to the 3' end of the non-coding strand template to complement the cohesive ends formed after digestion with BbsI. Three pairs of CRISPR oligonucleotide chains were designed, as shown in Table 1.

[0054] Table 1 FTO targeting sites and sgRNA oligonucleotide sequences

[0055]

[0056] 1.2 Vector construction

[0057] 1.21 Use BbsI to digest 1 μg of PX458 plasmid (purchased from Addgene), 30min, 37°C:

[0058]

[0059] 1.22 U...

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Abstract

The invention discloses a targeted-FTO-gene-knockout sgRNA (small guide ribonucleic acid) and CRISP (clustered regularly interspaced short palindromic repeats) / Cas9 slow virus system and application thereof. The sgRNA is selected from FTOsgRNAsp2 or FTOsgRNAsp3 with the following DNA sequence. The sgRNA has high cutting efficiency for the FTO gene. When the sgRNA-containing CRISPR / Cas9 slow virus system is used for transfecting SV-HUC-1, the FTO protein expression level of the obtained cell strain is obviously lowered. Therefore, the sgRNA disclosed by the invention can effectively implement targeted FTO gene knockout; and after the sgRNA is established into the CRISPR / Cas9 slow virus system, the system can knock out the FTO gene to obtain the FTO-gene-knockout cell strain, thereby being beneficial to researching the action mechanism of FTO in the cell strain.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a sgRNA targeted to knock out the FTO gene and a CRISPR / Cas9 lentivirus system and application. Background technique [0002] The FTO gene is an allele associated with obesity, also known as the obesity gene. The FTO gene is located on chromosome 16 (16q12.2), contains 9 exons, and has a gene length of 410.50kb. It is widely expressed in various developmental stages of human tissues, and is highly expressed in tissues such as hypothalamus, skeletal muscle and adipose tissue. The FTO gene is involved in the energy metabolism of cells. Because tumors are caused by excessive cell proliferation, and their energy metabolism is different from normal cells, it is necessary to study the mechanism of action of FTO in different cells. [0003] Bladder cancer is the most common urothelial tumor in my country's urology department. The vast majority originate from epithelial tissue, of ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N15/66C12N5/10
CPCC12N9/0071C12N15/1137C12N15/66C12N15/86C12N2310/10C12N2740/15043C12N2800/107C12N2800/80C12N2810/10C12Y114/11033
Inventor 纪卫东苏甲林阙彪张海青王敏
Owner THE FIRST AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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