Exogenous gene knocking-in and integrating system on basis of CRISPR/Cas9, method for establishing exogenous gene knocking-in and integrating system and application thereof

Abstract

The invention provides an exogenous gene knocking-in and integrating system on the basis of CRISPR / Cas9, a method for establishing the exogenous gene knocking-in and integrating system and application thereof. The exogenous gene knocking-in and integrating system comprises vectors with report / donor functions and Cas9 expression vectors. Each report / donor vector comprises two target gent homologous arms and an exogenous sequence fragment positioned between the two target gene homologous arms; homologous sequences, which are positioned on a target gene, of the two target gene homologous arms of each report / donor vector are respectively positioned on two sides of a target sequence of the target gene and are connected with the target sequence of the target gene; the exogenous sequence fragments comprise promoters, resistant genes, shorn peptide sequences, report genes and polyA tails which are sequentially arrayed, two SSA repair homologous sequences of each resistant gene are inserted into the resistant gene, and the target sequence of each target gene is inserted in a space between the two corresponding SSA repair homologous sequences. The exogenous gene knocking-in and integrating system, the method and the application have the advantages that exogenous genes can be integrated with endogenous gene sequences in an efficient site-directed and accurately targeted manner, and double-chromosome allelic gene double-knocking-in can be efficiently carried out.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to the knock-in and integration of exogenous genes mediated by CRISPR / Cas9 into endogenous alleles, and specifically relates to the application of genetic engineering technology to integrate exogenous functional genes into specific gene sites in animal cells to obtain Stably inherited transgenic cell lines. Background technique [0002] Site-directed gene editing is an important means to study gene function. This method is currently widely used in the study of animal and plant functional genes, the treatment and research of human diseases, and the production of transgenic animals and plants. Gene targeted modification is mainly based on gene targeting and DNA repair mechanism. DNA repair mechanisms include non-homologous end joining (NHEJ) and homologous recombination (HDR). In animal cells, NHEJ is the main repair method, and precise The gene editing is mainly based on t...

AI Technical Summary

Problems solved by technology

[0004] Although CRISPR / Cas9-mediated genome modification has become operational, precise genome editing still requires the use of HDR repair mechanisms, which are less efficient in animal cells.

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