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

A technology for exogenous genes and establishing methods, applied in the field of genetic engineering, can solve problems such as low efficiency of HDR mechanism

Active Publication Date: 2016-12-07
成都中科奥格生物科技有限公司
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Problems solved by technology

[0004] Although CRISPR/Cas9-mediated genome modification has become operational, precise genome e

Method used

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  • 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
  • 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
  • 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

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Embodiment Construction

[0046] In order to facilitate the understanding of the technical solution of the present invention, the method of knocking in exogenous genes through the CRISPR / Cas9-based double-knock-in system of the present invention will be further described in conjunction with specific examples targeting the CCR5 gene (Genbank accession number NC_000003.12) below .

[0047] Table I. Primers required for construction of double knockout system

[0048]

[0049]

[0050] In Table I, the restriction endonucleases corresponding to the restriction endonucleases marked in parentheses after the primer names are indicated in italics in the sequence. CCR5 target sequences contained in CCR5a annealing f and CCR5a annealing r are underlined.

[0051] (1) Reporter and donor system vector pXL-L / R.CCR5 arm-CAG-PuroR(200bp repeat.SSA-CCR5)-T2A-eGFP(Rep / Don PG) and pXL-L / R.CCR5 arm-CAG - Construction of ZeoR (200bp repeat.SSA-CCR5)-T2A-RFP (Rep / Don ZR).

[0052] (1) Fit the primers CCR5a annealin...

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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...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N5/10
CPCC12N15/66C12N15/85C12N2800/107C12N2800/80
Inventor 张智英吴芸徐坤白义春吕慧娇
Owner 成都中科奥格生物科技有限公司
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