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Application of Differential Agent Technology in Cell Enrichment of a·g Base Substitution

A base and cell technology, applied in the application field of differential surrogate technology in the enrichment of A·G base replacement cells, to achieve the effect of improving replacement efficiency and simple technical design

Active Publication Date: 2021-07-16
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are very limited studies on the enrichment of A·G base replacement cells by reporter gene-mediated cell enrichment technology in plants, and there is no use of selection markers during transformation to achieve A·G base replacement at the cellular level. Enrichment of cells to improve the efficiency of A·G base substitution

Method used

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  • Application of Differential Agent Technology in Cell Enrichment of a·g Base Substitution
  • Application of Differential Agent Technology in Cell Enrichment of a·g Base Substitution
  • Application of Differential Agent Technology in Cell Enrichment of a·g Base Substitution

Examples

Experimental program
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Effect test

Embodiment 1

[0137] Example 1, Establishment of EcTadA&ecTadA&Cas9n-mediated differential proxy technology for A G base substitution

[0138] 1. Establishment of EcTadA&ecTadA&Cas9n-mediated differential agent technology carrier for A·G base substitution

[0139] The common technical carrier of EcTadA&ecTadA&Cas9n(ABE)-mediated A G base replacement was named sgRNA-GT.

[0140] The carrier of differential agent technology for EcTadA&ecTadA&Cas9n(ABE)-mediated A·G base substitution was named DisSUGs.

[0141] The schematic diagrams of sgRNA-GT and DisSUGs vectors are as follows figure 1 shown.

[0142] The difference between the differential agent technology carrier and the ordinary technology carrier is:

[0143] 1) Differential proxy technology carrier transforms the screening agent resistance gene in the common technology carrier to lose its function, and adds the corresponding proxy target sequence to the sgRNA part. Taking the selection agent resistance gene as hygromycin resistanc...

Embodiment 2

[0147] Example 2, Construction of EcTadA&ecTadA&Cas9n-mediated differential agent technology carrier and its application in rice genome editing

[0148] 1. Construction of recombinant expression vector

[0149] The recombinant expression vectors in this example are divided into the following two types: DisSUGs recombinant expression vectors and sgRNA-GT recombinant expression vectors. The structural diagrams of the components of the two recombinant expression vectors are as follows: figure 1 shown. Each vector is a circular plasmid.

[0150] According to the different target sequences contained, each recombinant expression vector is divided into three types, and there are six recombinant expression vectors as follows: DisSUGs-1 recombinant expression vector, DisSUGs-2 recombinant expression vector, DisSUGs-3 recombinant expression vector, sgRNA-GT -1 recombinant expression vector, sgRNA-GT-2 recombinant expression vector, sgRNA-GT-3 recombinant expression vector.

[0151]...

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Abstract

The invention discloses the application of the differential agent technology in the enrichment of A·G base substitution cells. The differential agent technology carrier of the present invention includes esgRNA targeting target gene target sequence, sgRNA targeting function loss screening agent resistance gene target sequence, A.G base substitution system and function loss screening agent resistance gene ; The A·G base replacement system is guided by the sgRNA targeting the target sequence of the screening agent resistance gene for loss of function, and can perform A·G base replacement on the target sequence of the screening agent resistance gene for the loss of function. Replacement restores the function of the loss-of-function selection agent resistance gene. The invention realizes the enrichment of A·G base substitution cells at the cell level, and greatly improves the efficiency of A·G base substitution.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of differential agent technology in A·G base substitution cell enrichment. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), and cuts to generate a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ) and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base substitution...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N15/66C12N15/82C12N9/22
CPCC12N9/22C12N15/65C12N15/66C12N15/8209C12N15/8218
Inventor 杨进孝赵久然张成伟徐雯武莹吕欣欣
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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