Method for efficiently carrying CRISPR/Cas9 through functionalized graphene oxide for gene editing

Abstract

The invention discloses a method for efficiently carrying CRISPR/Cas9 through functionalized graphene oxide for gene editing. An NGO-PEG-PEI/Cas9/sgRNA complex is obtained by the following steps: covalently modifying aminated PEG and aminated PEI onto graphene oxide to obtain NGO-PEG-PEI; then assembling Cas9 protein and sgRNA into a Cas9/sgRNA complex under a room-temperature condition; and mixing NGO-PEG-PEI with the Cas9/sgRNA complex under a room-temperature condition. The editing of target gene can be realized by mixing the complex with the target gene. The functionalized GO has the characteristics of good biocompatibility and high carrying efficiency, can efficiently carry the Cas9/sgRNA complex into cells for performing corresponding functions, and has the effect of keeping the Cas9/sgRNA complex free of enzymatic degradation and high in stability.

Description

Technical field [0001] The invention belongs to the technical field of biological detection, and particularly relates to a method for efficiently carrying CRISPR / Cas9 on functionalized graphene oxide for gene editing. Background technique [0002] CRISPR (Clustered regularly interspaced short palindromic repeats) is an adaptive immune response system found in bacteria, which can effectively resist the damage caused by invading viruses and foreign DNA. The CRISPR / Cas9 system modified from the type II CRISPR system consists of single-stranded sgRNA and Cas9 protein with endonuclease activity. The sgRNA guides Cas9 nuclease to cut at a specific DNA sequence to produce double-stranded end breaks. Cells can Repair by HDR or NHEJ. As a breakthrough research technique in the field of life sciences, CRISPR / Cas9 technology has been widely used in gene editing, regulation of gene expression, genome screening, visualization of genome dynamics, and imaging of RNA in mammalian living cells. ...

AI Technical Summary

Problems solved by technology

Cell-penetrating peptide (CPP) forms a covalently linked complex with Cas9 nuclease in the form of thioether bonds, and transports Cas9 protein and sgRNA into cells by electrostatic adsorption with sgRNA, and is used in endogenous human cell lines However, in this method, the Cas9 protein and sgRNA are not transported into the cell in the form of an assembled complex, resulting in low gene editing efficiency

A method based on cationic liposomes has also been developed to transport Cas9 / sgRNA complexes into cells to edit the genome, but the instability of cationic liposomes has brought inconvenience to the transport of Cas9 / sgRNA complexes; DNA nano Spheres (NCs) are also widely used as cell transport carriers because of their low toxicity, but NCs are formed from exogenous DNA, which may bring about new immune responses after carrying Cas9 / sgRNA into cells; Cas9En-ArgNP nanocomposite Although the drug can deliver Cas9 / sgRNA into the cytoplasm, its toxicity to cells needs further verification

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