Cell enrichment technology and its application of c·t base substitution using mutated screening agent resistance gene as reporter system

A technology of resistance gene and screening agent, applied in application, genetic engineering, recombinant DNA technology, etc., to achieve simple technical design and improve the efficiency of C T base replacement

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

AI Technical Summary

Problems solved by technology

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

Method used

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  • Cell enrichment technology and its application of c·t base substitution using mutated screening agent resistance gene as reporter system
  • Cell enrichment technology and its application of c·t base substitution using mutated screening agent resistance gene as reporter system
  • Cell enrichment technology and its application of c·t base substitution using mutated screening agent resistance gene as reporter system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Example 1, Establishment of Cell Enrichment Technology for C T Base Substitution

[0161] 1. Establishment of cell enrichment technology carrier for C T base substitution

[0162] The common technology (non-cell enrichment technology) carrier of Cas9 nuclease, cytosine deaminase and UGI-mediated C T base replacement was named sgRNA-GT.

[0163] The cell enrichment technology carrier of Cas9 nuclease, cytosine deaminase and UGI-mediated C T base replacement was named sgRNA -TP -Hyg -TP / sgRNA-GT.

[0164] Take the Cas9 nuclease as SpCas9n and the cytosine deaminase as PmCDA1 as an example: sgRNA -TP -Hyg -TP The schematic diagram of the structure of / sgRNA-GT and sgRNA-GT carrier is as follows figure 1 shown.

[0165] The cell enrichment technology carrier is to mutate the screening agent resistance gene on the basis of the non-cell enrichment technology carrier to make it lose its function, and at the same time add the corresponding target sequence (surrogate tar...

Embodiment 2

[0170] Example 2, Construction of Cas9n&PmCDA1&UGI-mediated cell enrichment technology vector and its application in rice genome editing

[0171] 1. Construction of recombinant expression vector

[0172] The recombinant expression vectors in this example are the general technology carrier sgRNA-GT for Cas9n&PmCDA1&UGI (PCBE)-mediated C T base replacement and the cell enrichment technology carrier sgRNA for Cas9n&PmCDA1&UGI (PCBE)-mediated C T base replacement -TP -Hyg -TP / sgRNA-GT. Each vector is a circular plasmid. The structural diagrams of the components of the two recombinant expression vectors are as follows: image 3 shown.

[0173] According to the different target sequences contained, each recombinant expression vector is divided into two types, and there are four recombinant expression vectors as follows: sgRNA -TP -Hyg -TP / sgRNA-GT-1 recombinant expression vector, sgRNA -TP -Hyg -TP / sgRNA-GT-2 recombinant expression vector, sgRNA-GT-1 recombinant expressio...

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Abstract

The invention discloses a C·T base replacement cell enrichment technology and its application using a mutant screening agent resistance gene as a reporter system. The cell enrichment technology includes the following reagents: sgRNA, C T base replacement system and screening agent resistance gene for loss of function; sgRNA consists of tRNA-sgRNA targeting the target gene target sequence and screening agent for targeting loss The tRNA-sgRNA composition of the resistance gene target sequence; the screening agent resistance gene with loss of function is the sequence obtained after the screening agent resistance gene is subjected to non-functional mutation; The C·T base substitution of the target sequence at the locus can restore the function of the screening agent resistance gene that has lost its function. The invention realizes the enrichment of C·T base replacement cells at the cell level, and greatly improves the efficiency of C·T base replacement.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a C·T base replacement cell enrichment technology using a mutant screening agent resistance gene as a reporter system and its application. 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 substitution, the applicat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/82A01H5/00A01H6/46
CPCC12N15/8218C12N15/902
Inventor 杨进孝杨永星赵思李璐袁爽
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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