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Method for improving plant gene replacing efficiency

A gene replacement and plant technology, applied in botany equipment and methods, biochemical equipment and methods, plant products, etc.

Active Publication Date: 2020-04-03
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in plants, there are very few reports on the use of P2A to couple target proteins with other marker proteins

Method used

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  • Method for improving plant gene replacing efficiency
  • Method for improving plant gene replacing efficiency
  • Method for improving plant gene replacing efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1. Construction of a carrier for gene replacement without DNA double-strand breaks and its application in rice gene replacement

[0098] 1. Construction of recombinant expression vector and description of replacement principle

[0099] 1. Construction of recombinant expression vector

[0100] Artificially synthesize the following recombinant expression vectors, each of which is a circular plasmid: sgRNA / Cas9 recombinant expression vector, sgRNA / Cas9n recombinant expression vector. The structural schematic diagrams of sgRNA / Cas9 recombinant expression vector and sgRNA / Cas9n recombinant expression vector are as follows: figure 1 shown. The specific structure description is as follows:

[0101] The sequence of the sgRNA / Cas9n recombinant expression vector is sequence 1 in the sequence list. The 131-467th position of sequence 1 is the OsU3 promoter sequence, the 474-550th position is the tRNA sequence, the 551-570th position is the ST215 target sequence, the 571...

Embodiment 2

[0145] Example 2, Optimization of sgRNA / Cas9n recombinant expression vector and its application in rice gene replacement

[0146] 1. Construction of recombinant expression vector and description of replacement principle

[0147] 1. Construction of recombinant expression vector

[0148] Artificially synthesize the following recombinant expression vectors, each of which is a circular plasmid: sgRNA / Cas9n recombinant expression vector, esgRNA / Cas9n-P2A-Hpt recombinant expression vector. Schematic diagram of the structure of sgRNA / Cas9n recombinant expression vector and esgRNA / Cas9n-P2A-Hpt recombinant expression vector Figure 7 shown. The specific structure description is as follows:

[0149] The sequence of the sgRNA / Cas9n recombinant expression vector is sequence 1 in the sequence list.

[0150] The sequence of the esgRNA / Cas9n-P2A-Hpt recombinant expression vector is to replace the 571-646 in the sequence 1 with the esgRNA backbone sequence shown in the sequence 7, and re...

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Abstract

The invention discloses a method for improving plant gene replacing efficiency. The method comprises the following steps of guiding esgRNA, a Cas9 nicking enzyme, a screening agent resistant protein and donor DNA into a purpose plant, enabling the esgRNA to target a DNA fragment A target point sequence, performing coexpression on the Cas9 nicking enzyme and the screening agent resistant protein, wherein the donor DNA sequentially comprises the DNA fragment A target point sequence, a DNA fragment B and a DNA fragment A target point sequence, the DNA fragment B is DNA molecules obtained throughperforming mutation of one or several basic groups on the DNA fragment A, under the guidance of the esgRNA, the Cas9 nicking enzyme produces single-stranded DNA nicking at the DNA fragment A target point sequence in the purpose plant genome and the DNA fragment A target point sequence in the donor DNA, through an in vivo repair mechanism of the purpose plant, the DNA fragment A in the purpose plant genome is replaced with the DNA fragment B, so that plant gene replacement is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for improving plant gene replacement efficiency. Background technique [0002] The probability of precise gene replacement mediated by long-strand DNA templates in cells is very low, but introducing a DNA double-strand break (dsDNA break, DSB) near the site to be replaced can significantly increase the probability of replacement. CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9protein-RNA complex is positioned on the target by the guide RNA (guide RNA), and the DNA is cleaved to generate DSBs, thereby increasing the efficiency of precise gene replacement mediated by long-strand DNA templates. After the generation of DSB, the organism will instinctively start the DNA repair mechanism. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), which acc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/65C12N9/22A01H5/00A01H6/46
CPCC12N9/1022C12N9/22C12N15/113C12N15/65C12N15/8218C12Y202/01006C12N2310/20
Inventor 徐雯武莹杨进孝宋伟杨永星贺晓庆
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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