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Technology for enriching A.G base-substituted cells by taking inactivated screening agent resistance gene a reporting system and application thereof

A technology of resistance gene and screening agent, applied in the field of cell enrichment for A·G base substitution

Active Publication Date: 2019-12-31
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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  • Technology for enriching A.G base-substituted cells by taking inactivated screening agent resistance gene a reporting system and application thereof
  • Technology for enriching A.G base-substituted cells by taking inactivated screening agent resistance gene a reporting system and application thereof
  • Technology for enriching A.G base-substituted cells by taking inactivated screening agent resistance gene a reporting system and application thereof

Examples

Experimental program
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Embodiment 1

[0132] Example 1. Establishment of cell enrichment technology for EcTadA&ecTadA&Cas9n-mediated A G base substitution

[0133] 1. The establishment of the cell enrichment technology vector for EcTadA&ecTadA&Cas9n-mediated A·G base substitution

[0134] The general technology (non-cell enrichment technology) carrier of EcTadA&ecTadA&Cas9n(ABE)-mediated A G base replacement was named sgRNA-GT.

[0135] The cell enrichment technology carrier of EcTadA&ecTadA&Cas9n(ABE)-mediated A·G base replacement was named sgRNA -ATG -Hyg -ATG / sgRNA-GT.

[0136] sgRNA-GT and sgRNA -ATG -Hyg -ATG The schematic diagram of the structure of / sgRNA-GT vector is as follows figure 1 shown.

[0137] The cell enrichment technology carrier is a carrier obtained by modifying the screening agent resistance gene on the basis of the non-cell enrichment technology carrier to lose its function, and adding the corresponding proxy target to the sgRNA part.

[0138] Taking the selection agent resistance g...

Embodiment 2

[0141] Example 2. Construction of EcTadA&ecTadA&Cas9n-mediated cell enrichment technology vector and its application in rice genome editing

[0142] 1. Construction of recombinant expression vector

[0143] The recombinant expression vectors in this embodiment are divided into the following two types: sgRNA -ATG -Hyg -ATG / sgRNA-GT recombinant expression vector, sgRNA-GT recombinant expression vector. The schematic diagrams of the two recombinant expression vectors are shown in image 3 shown. Each vector is a circular plasmid.

[0144] 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: sgRNA -ATG -Hyg -ATG / sgRNA-GT-1 recombinant expression vector, sgRNA -ATG -Hyg -ATG / sgRNA-GT-2 recombinant expression vector, sgRNA -ATG -Hyg -ATG / sgRNA-GT-3 recombinant expression vector, sgRNA-GT-1 recombinant expression vector, sgRNA-GT-2 recombinant ...

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Abstract

The invention discloses a technology for enriching A.G base-substituted cells by taking an inactivated screening agent resistance gene as a reporting system and application thereof. A cell enrichmenttechnology vector comprises the following reagents: sgRNA of a target sequence of a target gene, sgRNA of a target sequence of a screening agent resistance gene with target function loss, an A.G basesubstitution system and a screening agent resistance gene with function loss. Under the guidance of the sgRNA of the target sequence of the screening agent resistance gene with target function loss, the A.G base substitution system can carry out A.G base substitution on the target sequence of the screening agent resistance gene with function loss so as to restore the function of the screening agent resistance gene with function loss. The technology realizes the enrichment of A.G base-substituted cells at the cell level and greatly improves the A.G base substitution efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a cell enrichment technology and application thereof for A·G base substitution using an inactivated screening agent resistance gene as a reporter system. 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), cuts and generates 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 substitutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/66C12N15/82C12N9/22
CPCC12N15/65C12N15/66C12N15/8209C12N15/8218C12N9/22
Inventor 杨永星杨进孝康桂婷王飞鹏宋金岭
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES