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

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...

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

Images