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Gene editing tool, preparation method thereof and multi-round gene editing method

A gene editing and tool technology, applied in the field of genetic engineering, can solve the problem of time-consuming and laborious multi-round gene editing methods

Active Publication Date: 2020-10-30
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem mainly solved by the present invention is to provide a multi-round gene editing method without plasmid elimination, which can solve the time-consuming and labor-intensive problems of existing multi-round gene editing methods (requiring additional screening marker removal or plasmid elimination steps)

Method used

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  • Gene editing tool, preparation method thereof and multi-round gene editing method
  • Gene editing tool, preparation method thereof and multi-round gene editing method
  • Gene editing tool, preparation method thereof and multi-round gene editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. The preparation method of the gene editing tool of the present invention.

[0088] The gene editing tools described in this example include the following three sgRNA expression plasmids, namely the pRock series mother plasmids, the pPaper series mother plasmids and the pScissors series mother plasmids, wherein each mother plasmid contains, in addition to expressing the target gene for editing. In addition to the sgRNA framework, it also contains sgRNA that targets one of the other two parent plasmids; in the process of gene editing, the gene editing plasmids constructed from the above three parent plasmids are recycled and used first in the host cell. The plasmid or selection marker is eliminated by the gene editing plasmid used later. The following example describes its preparation method:

[0089] 1. Construction of the common template plasmid pRPS of pRock, pPaper and pScissors.

[0090] 1. Amplify the sgRNA-curing frame part.

[0091] The whole gene syn...

Embodiment 2

[0114] Embodiment 2. pRock, pPaper, pScissors mutual elimination result test.

[0115] 1. pPaper eliminates pRock tests.

[0116] (1) Transform pCas and pRock series master plasmids into W3110 strain to obtain strain CasR.

[0117] (2) Transform the pPaper series parent plasmids into CasR, take the same amount of transformation products and apply them on Kan+Cm and Kan+Cm+Tet plates, respectively, and compare the elimination effect of pPaper and pRock. Such as image 3 As shown, pPaper can effectively eliminate pRock.

[0118] (3) Transform the pRPS-C obtained in Example 1 into CasR, and apply equal amounts of the transformation products to Kan+Cm and Kan+Cm+Tet plates respectively, and compare the elimination effect without gRNA for curing, that is, the negative control. Such as image 3 It was shown that compared with pPaper, pRPS-C had a low conversion efficiency and could not effectively eliminate pRock, and the efficiency of eliminating pRock was 26.07±27.02%.

[011...

Embodiment 3

[0130] Example 3 Construction of pRock, pPaper, pScissors series of plasmids.

[0131] The gene editing tools pRock, pPaper, and pScissors series master plasmids prepared by the present invention can be widely used in editing experiments of target genes. In this example, the genes related to the pyruvate synthesis pathway are used as examples to describe the gene editing prepared by the present invention. A method for constructing gene-editing vectors.

[0132] Specific pRock, pPaper, pScissors series of plasmids according to figure 2 design. After the system construction is complete, the new plasmid only needs to be constructed from the mother plasmid obtained in Example 1, amplified in four fragments, and primer design and plasmid construction are carried out according to the instructions of the in vitro multi-fragment recombination kit. The process is as follows: Figure 6 shown. The following three series are respectively illustrated with examples.

[0133] 1. Constru...

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Abstract

The invention discloses a gene editing tool, a preparation method thereof and a multi-round gene editing method, and belongs to the technical field of gene engineering. The problem that an existing multi-round gene editing method wastes time and labor is solved. The invention provides a gene editing tool, which comprises the following three sgRNA expression parent plasmids: pRock series mother plasmids, pPaper series mother plasmids and pScissors series mother plasmids, wherein each mother plasmid can express sgRNA used for editing a target gene and can also express sgRNA targeting one of theother two mother plasmids. In the process of implementing gene editing, when the gene editing plasmids constructed by the three mother plasmids are recycled, the gene editing plasmids which are firstly used in host cells or the selection markers are eliminated by the gene editing plasmids which are later used. According to the method, the editing period is shortened, the multi-round gene editing speed is increased, and the possibility of spontaneous mutation of the genome can be reduced due to fewer culture times.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene editing tool, a preparation method thereof, and a method for multiple rounds of gene editing. Background technique [0002] Gene editing has been widely applied to the transformation of various species, and has been widely used in basic scientific research and applied research. For example, through gene editing, a large number of strains that meet different needs have been successfully constructed. The classic bacterial gene editing method is accomplished through homologous recombination mediated by phage-derived recombinases (such as λRed, RecET). This approach generally involves two steps. In the first step, homologous fragments are recombined into specific genomic locations, but due to the low recombination efficiency, selection markers must be introduced at this step. In the second step, the selection marker in the first step is removed, ther...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/70C12N15/66C12P7/40C12R1/19
CPCC12N15/902C12N15/70C12N15/66C12P7/40
Inventor 赵广咸漠王纪超隋新悦
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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