CRISPR/Cas9 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof

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

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

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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 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof
  • CRISPR/Cas9 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof
  • CRISPR/Cas9 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof

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Embodiment 1

[0044] 1. Construction of knockout RITA plasmids using CRISPR / Cas9 technology

[0045] 1.1 sgRNA oligonucleotide chain synthesis

[0046] Use the CRISPR online design tool (http: / / crispr.mit.edu / ) to design a 20bp sgRNA on the second exon of RITA according to the scoring system, and verify that there are no non-specific genes by BLAST. ACCG is added to the 5′ end of the coding strand template, and AAAC is added to the 3′ end of the non-coding strand template, which is complementary to the cohesive end formed after digestion with BsmBI, and a pair of CRISPR oligonucleotide chains are designed, see Table 1, Table 1 RITA targeting position Dot and sgRNA oligonucleotide sequence.

[0047]

[0048] 1.2 Vector construction

[0049] 1.2.1 Use BsmBI to digest 2 μg Lenti-CRISPRv2 plasmid (purchased from Addgene), 2h, 37°C,

[0050] The enzyme digestion system is:

[0051]

[0052] 1.2.2 Purify the enzyme-digested plasmid product with Jerry Gene Gel Recovery Kit, and operate a...

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Abstract

The present invention discloses CRISPR/Cas9 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof. First, sgRNA specifically targeted to a second exon of the RITA gene isobtained, and the base sequence of the sgRNA is as shown in SEQ ID NO. 1; secondly, a sgRNA lentiviral vector system of the RITA gene is constructed, and the sgRNA lentiviral vector system contains Cas9 protein; and finally human intestinal cancer HT-29 cells are infected with CRISPR/Cas9 lentivirus containing the sgRNA to obtain a cell line which is significantly reduced in RITA protein expression level. The invention has the advantages of simple operation steps, good sgRNA targeting property, and high RITA gene cutting efficiency; furthermore, the constructed CRISPR/Cas9 lentivirus system has the advantage of high knock-out efficiency and can specifically knock out the RITA gene to obtain RITA gene-knockout human intestinal cancer cells, and a powerful tool for the further study of theaction mechanism of RITA in the 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 RITA gene and its specific sgRNA. Background technique [0002] RBP-J interacting and microtubule-associated protein (RITA; C12orf52) is a highly conserved protein with a protein size of 36kD and no significant homology to any other protein. RITA can interfere with Notch and RBP-J-mediated transcription. RITA rapidly shuttles between the nucleus and cytoplasm, and most importantly, it mediates nuclear export of RBP-J tubulin fibers. RITA, as a novel RBP-J interacting protein, down-regulates Notch by interfering with the transcription factor RBP-J. The Notch genes encode a class of highly conserved cell surface receptors that regulate cell development in organisms ranging from sea urchins to humans. Notch signaling affects multiple processes in normal cell morphogenesis, including the specification ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10C12N7/01
CPCC12N5/0693C12N7/00C12N15/1135C12N15/86C12N2310/10C12N2510/00C12N2740/15022C12N2740/15043C12N2800/80C12N2810/10
Inventor 高玉涛杨佳丽杨兴林潘讴东
Owner OBIO TECH SHANGHAI CORP LTD
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