Gene editing system based on CRISPR-Cas9 technology and application of gene editing system

Abstract

The invention provides a gene editing system based on a CRISPR-Cas9 technology and application of the gene editing system. Based on an endogenous U6 promoter dependent-form vector system, a negative selection marker is added into a Cas9 expression box of an expression vector, an anti-drug selection marker on a rescue vector containing a homologous arm is transferred onto the expression vector, thus the base size of the rescue vector is decreased, and the capacity of the rescue vector is increased; and an exogenous gene segment lengthening out to 6.3 kb can be input in human plasmodium in a mediated mode, obtained recombinant plasmodium does not contain the anti-drug selection marker and Cas9 protein expression plasmid residue, continuous gene editing can be conducted through the same drugselection marker, and the gene editing system is stable in performance, efficient, concise, and powerful in function, and has wide application prospects and huge market value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a gene editing system and application based on CRISPR-Cas9 technology. Background technique [0002] Gene editing is a technology that uses artificial nucleases or "molecular scissors" to insert, delete or replace DNA, including the first-generation ZFN zinc finger nuclease technology, the second-generation TALEN technology and the third-generation CRISPR / Cas9 technology . CRISPR / Cas9 technology has the advantages of simple design, multiple sites, high efficiency, good specificity, and short time-consuming. It is currently the hottest gene editing technology. The main components of the CRISPR / Cas9 system are sgRNA (single guide RNA) targeting the target gene and Cas9 nuclease. The working principle of this system is that Cas9 nuclease, under the guidance of sgRNA, cuts at a specific site of the target gene to generate a gap. After the gap is generated, cells can repair it in a varie...

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Problems solved by technology

[0004] The disadvantages of these two systems are: 1. The worms after gene editing contain drug resistance selection markers and residues of Cas9 protein expression vectors

At present, the resistance screening markers available in Pf are very limited, only hdhfr, bsd (blasticidin Sdeaminase), neo (neomycin phosphotransferase), pac (puromycin-N-acetyltransferase), and ydhodh (yeast dihydroorotate dehydrogenase) are available for selection, and Drugs are expensive, and some of them have not been commercialized; if multiple genes of Plasmodium need to be edited, the above two gene editing systems may be difficult to achieve multiple rounds of gene editing for Pf due to insufficient drug screening markers; and the Cas9 protein Residues of the expression vector will cause potential off-target risk of gene editing; 2. The size of the plasmid carrying the homology arm for homology repair is relatively large, and it is difficult to load large fragments for knock-in operation

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