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Fusion protein and design method thereof

A fusion protein and protein technology, applied in the biological field, can solve the problems of low efficiency of recombinant gene editing and less recombination repair, and achieve the effect of improving the efficiency of recombination repair

Inactive Publication Date: 2020-08-11
重庆英茂盛业生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In animal cells, the repair method after genomic DNA breakage is mainly adjacent-end junction repair, and the proportion of recombination repair is very small, resulting in low efficiency of recombination gene editing, and it is necessary to screen a large number of clones to obtain correctly edited cells

Method used

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  • Fusion protein and design method thereof

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Experimental program
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Effect test

Embodiment 1

[0025] Fusion protein 1: design method of spCas9-Rad51(H) and expression and purification of spCas9-Rad51(H) protein with E. coli expression host.

[0026] Fusion protein 1: design method of spCas9-Rad51(H).

[0027] 1. The protein functional domain with specific DNA cutting function selects spCas9 protein;

[0028] 2. Human Rad51 protein is selected as the recombinant auxiliary protein functional domain with DNA binding function;

[0029] 3. The two are connected with polypeptide GGGGSGGSGGSGGGS;

[0030] 4. The protein structure is spCas9-connecting peptide-Rad51(H).

[0031] The method of fusion expressing spCas9-Rad51(H) using Escherichia coli prokaryotic expression system, the realization steps are as follows:

[0032] 1. Connect the artificially synthesized spCas9-Rad51(H) coding sequence to the prokaryotic expression vector pET-30a to obtain the expression vector pET-spCas9-Rad51(H);

[0033] 2. Transform Escherichia coli expression strain BL21(DE3), the transformat...

Embodiment 2

[0048] Fusion protein 2: the design method of spCas9-Rad52(H), the specific implementation steps are as follows:

[0049] 1. The protein functional domain with specific DNA cutting function selects spCas9 protein;

[0050] 2. Human Rad52 protein is selected as the recombinant auxiliary protein functional domain with DNA binding function;

[0051] 3. The two are connected with polypeptide GGGGSGGSGGSGGGS;

[0052] 4. The protein structure is spCas9-connecting peptide-Rad52(H).

[0053] The expression and purification methods of spCas9-Rad52(H) protein are the same as in Example 1.

Embodiment 3

[0055] Fusion protein 3: The design method of Rad51(H)-spCas9, the specific implementation steps are as follows:

[0056] 1. The protein functional domain with specific DNA cutting function selects spCas9 protein;

[0057] 2. Human Rad51 protein is selected as the recombinant auxiliary protein functional domain with DNA binding function;

[0058] 3. The two are connected with polypeptide GGGGSGGSGGSGGGS;

[0059] 4. The protein structure is Rad51(H)-linked peptide-spCas9.

[0060] The expression and purification methods of Rad51(H)-spCas9 protein are the same as in Example 1.

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Abstract

The invention discloses a fusion protein and a design method thereof. The design method comprises the following steps: fusing a recombinant helper protein with the DNA binding function and a protein with the DNA cleavage function. When the fusion protein with the DNA cleavage function performs cleavage, the recombinant helper protein provides a DNA recombinant repair template near a cleavage site,and recruits recombinant repair enzyme, so that the recombinant repair efficiency is greatly improved. The fusion protein has the advantage that the recombination repair efficiency is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein and a design method thereof. Background technique [0002] Cas9 gene editing technology has been widely used in gene editing of various organisms and cells cultured in vitro. The current conventional method is that after the Cas9 protein cuts the genomic DNA at a specific point, the DNA breakpoint is usually repaired by the biological or cell's own repair system. Repair methods include imprecise adjacent-end joining repair and precise recombination repair. In animal cells, the repair method after genomic DNA breakage is mainly adjacent-end junction repair, and the proportion of recombination repair is very small, resulting in low efficiency of recombination gene editing, and it is necessary to screen a large number of clones to obtain correctly edited cells. [0003] Therefore need a kind of Cas9 fusion protein and the method for designing similar protein. A simil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C07K14/47C12N9/22
CPCC07K14/4702C07K2319/80C12N9/22
Inventor 桂有静
Owner 重庆英茂盛业生物科技有限公司
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