CRISPR-Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and specific sgRNA thereof

Abstract

The invention discloses a CRISPR / Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and specific sgRNA thereof. The CRISPR / Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and the specific sgRNA thereof are characterized in that: firstly, sgRNA of a second exon of the specific targeted DEAF1 gene is obtained and the base sequence of the sgRNA is shown as SEQ ID NO.1; secondly, the sgRNA of the DEAF1 gene is constructed into a lentiviral vector system, which contains Cas9 protein; finally, the CRISPR / Cas9 lentivirus containing the sgRNA is infected with human colorectal carcinoma cell HT-29 cell, so that a cell strain of which DEAF1 protein expression level is obviously reduced is obtained. The CRISPR / Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene disclosed by the invention has the advantages of simple operation steps, good sgRNA target ability and high cutting efficiency for the DEAF1 gene; in addition, the constructed CRISPR / Cas9 lentiviral vector system has the advantage of high knockout efficiency and can specifically knock out the DEAF1 gene to obtain the human colorectal carcinoma cells knocking out the DEAF1 gene, and therebya powerful tool is provided for further studying an action mechanism of DEAF1 in the colorectal carcinoma cells.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to CRISPR / Cas9 targeted knockout of the DEAF1 gene of human intestinal cancer cells and its specific sgRNA. Background technique [0002] RBP-J interacting and microtubule-associated protein (DEAF1; C12orf52) is a highly conserved protein with a protein size of 36 kD and no significant homology to any other protein. DEAF1 can interfere with Notch- and RBP-J-mediated transcription. DEAF1 rapidly shuttles between the nucleus and cytoplasm and, most importantly, mediates nuclear export of RBP-J tubulin fibrils. DEAF1, as a novel RBP-J interacting protein, downregulates Notch by interfering with the transcription factor RBP-J. Notch genes encode a class of highly conserved cell surface receptors that regulate the development of cells in a variety of organisms, from sea urchins to humans. Notch signaling affects multiple processes of normal cell morphogenesis, includ...

AI Technical Summary

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 tumor suppressor genes (point mutations, rearrangements, deletions) and chromosomal Abnormalities, etc., but the molecular mechanisms involved in the occurrence and progression of colorectal cancer are still unclear, and further research is needed

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