CRISPR-Cas9 for targeting knockout of human intestinal cancer cell CNR1 gene, and specific sgRNA thereof

A colorectal cancer cell-specific technology, applied in the field of gRNA, can solve the problem that the molecular mechanism of colorectal cancer has not yet been clarified

Inactive Publication Date: 2018-09-28
OBIO TECH SHANGHAI CORP LTD
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Problems solved by technology

At present, researchers have done a lot of research on the pathogenesis of colorectal cancer, and have discovered many pathways and mechanisms involved in the progression of colorectal cancer, such as activation of proto-oncogenes, inactivation of

Method used

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  • CRISPR-Cas9 for targeting knockout of human intestinal cancer cell CNR1 gene, and specific sgRNA thereof
  • CRISPR-Cas9 for targeting knockout of human intestinal cancer cell CNR1 gene, and specific sgRNA thereof
  • CRISPR-Cas9 for targeting knockout of human intestinal cancer cell CNR1 gene, and specific sgRNA thereof

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Embodiment 1

[0044] 1. Construction of a knockout CNR1 plasmid using CRISPR / Cas9 technology

[0045] 1.1 sgRNA oligonucleotide chain synthesis

[0046] Using the CRISPR online design tool (http: / / crispr.mit.edu / ) according to the scoring system, a 20bp sgRNA was designed on the second exon of CNR1, and no non-specific genes were verified by BLAST. ACCG 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 BsmBI, and a pair of CRISPR oligonucleotide chains were designed, as shown in Table 1.

[0047] Table 1 CNR1 targeting site and sgRNA oligonucleotide sequence

[0048]

[0049] 1.2 Vector construction

[0050] 1.2.1 Use BsmBI to digest 2 μg of Lenti-CRISPRv2 plasmid (purchased from Addgene) for 2 hours at 37°C. The enzyme digestion system is as follows:

[0051] 2μg (2μl)

Lenti-CRISPRv2

1μl

BsmBI (NEB)

5μl

10X Cutsmart

...

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Abstract

The invention discloses a CRISPR-Cas9 for targeting knockout of a human intestinal cancer cell CNR1 gene, and specific sgRNA sequences thereof. Firstly, an sgRNA specifically targeting CNR1 gene second exon is obtained, and base sequences of the sgRNA are as shown in SEQ ID NO. 1; secondly, the sgRNA of the CNR1 gene is constructed to a lentiviral vector system, and the lentiviral vector system contains Cas9 protein; and finally, a CRISPR/Cas9 lentivirus containing the sgRNA is infected with human intestinal cancer HT-29 cells, and a cell strain with CNR1 protein expression level significantlybeing reduced is obtained. The operation steps are simple, good sgRNA targeting is achieved, and the cutting efficiency to the CNR1 gene is high; and in addition, the constructed CRISPR/Cas9 lentiviral system has the advantage of high knockout efficiency, and can specifically knock out the CNR1 gene, human intestinal cancer cells with the CNR1 gene being knocked out is obtained, and therefore a powerful tool for further study of the mechanism of action of CNR1 in intestinal cancer cells is provided.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to CRISPR / Cas9 targeted knockout of human intestinal cancer cell CNR1 gene and its specific gRNA. Background technique [0002] The endocannabinoid system (Endocannabmd, EC) is a lipid signaling and neuroregulation system that has been studied in depth in recent years, and it is widely involved in the regulation of some physiological or pathological processes in the body. It is mainly composed of endocannabinoids, cannabinoid receptors, synthetases and degradative enzymes of endocannabinoids. In recent years, two cannabinoid receptors, CNR1 receptor and CNR2 receptor, have been discovered, both of which are G protein-coupled receptors. A large number of studies have shown that CNR1 is a key factor mediating the effects of endocannabinoids. The CNR1 receptor contains 473 amino acids and is mainly located in the brain, spinal cord and peripheral nervous system. The...

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10C12N15/90
Inventor 杨佳丽杨兴林刘梅锐潘讴东夏清梅
Owner OBIO TECH SHANGHAI CORP LTD
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