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CRISPR-Cas9 system-based gene editing vector and application thereof

A genome editing and vector technology, applied in genetic engineering, using vectors to introduce foreign genetic material, DNA/RNA fragments, etc., can solve problems such as affecting gene editing efficiency, improve editing efficiency, reduce transformation costs, and overcome low mutation rates Effect

Active Publication Date: 2017-09-15
BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression levels of Cas9 gene and sgRNA, as well as the sequence structure of sgRNA, will affect gene editing efficiency

Method used

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  • CRISPR-Cas9 system-based gene editing vector and application thereof
  • CRISPR-Cas9 system-based gene editing vector and application thereof
  • CRISPR-Cas9 system-based gene editing vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Selection of a promoter for driving the Cas9 gene

[0026] In this experiment, a nucleotide sequence on the wheat powdery mildew gene TaMLO-A gene was used as the target sequence, and the vector of the CRISPR-Cas9 system was optimized by comparing the gene editing efficiency of the CRISPR-Cas9 system vector when different promoters drove the expression of the Cas9 gene.

[0027] 1. Carrier Description

[0028] Both the p286 vector and the p294 vector contain two expression cassettes - sgRNA expression cassette and Cas9 expression cassette. The difference is that the promoter of the Cas9 expression cassette is different. The p286 vector uses the 2*35s promoter to drive the expression of the Cas9 gene, and the p294 vector uses ubi The promoter drives the expression of the Cas9 gene. Specifically:

[0029]The p286 vector includes TaU6p::TaMLO-A-sgRNA expression cassette and 2*35s::Cas9 expression cassette. TaU6p::TaMLO-A-sgRNA expression cassette elements inc...

Embodiment 2

[0038] Example 2 Selection of a promoter for driving sgRNA expression

[0039] In order to further improve the editing efficiency of wheat CRISPR-Cas9, we compared the editing efficiency of CRISPR-Cas9 vectors on wheat genome sequences when different snoRNA promoters drove the expression of sgRNA in wheat protoplast system. Currently plant The snoRNA promoters commonly used to drive the expression of sgRNA include TaU3p (SEQ ID NO: 10), OsU3p (SEQ ID NO: 11), TaU6 (SEQ ID NO: 1) and OsU6bp (SEQ ID NO: 12), which are also the focus of this experiment Several promoters for comparison.

[0040] 1. Carrier Description

[0041] p342 vector ( image 3 A), p338 vector ( image 3 B), p295 vector ( image 3 C) and p341 vector ( image 3 D) Both contain two expression cassettes——sgRNA expression cassette and Cas9 expression cassette. The Cas9 expression cassettes all include Ubi promoter (SEQ ID NO:7), Cas9 fragment (SEQ ID NO:5) and CaMV terminator (SEQ ID NO:6), and the target...

Embodiment 3

[0053] Example 3 Screening of different sequence structures of sgRNA

[0054] In the study of rice CRRISPR-Cas9 gene editing system, using tRNA transcription system to express sgRNA can increase the expression of sgRNA, thereby improving the gene editing efficiency of CRRISPR-Cas9; and in the study of human cells, it was found that the CRISPR-Cas9 system uses The gene editing efficiency varies greatly with different sgRNA sequence structures. In wheat, there are no studies and reports on the effect of sgRNA expression and its sequence structure on gene editing efficiency. In order to further optimize the vector of the wheat CRISPR-Cas9 system and improve the editing efficiency of the wheat genome sequence, we used the wheat genome sequence L475 as the target sequence (SEQ ID NO: 13) to target genes of CRISPR-Cas9 vectors containing different sgRNA sequence structures. Editing efficiency was compared.

[0055] 1. Carrier Description

[0056] p342 vector ( image 3 A), p345 ...

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Abstract

The invention provides a CRISPR-Cas9 system-based genome editing vector and an application thereof, belongs to the field of plant genetic engineering and particularly relates to a CRISPR-Cas9 system-based wheat genome editing vector and an application thereof. A CRISPR-Cas9 transformation vector with higher editing efficiency in wheat is obtained through comparing the gene editing efficiency of the transformation vector of a CRISPR-Cas9 system when adopting different Cas9 promoters, different sgRNA promoters and different sgRNA sequence structures. Through the optimized vector, the gene editing efficiency of the wheat crop by using CRISPR-Cas9 is improved; the problems of low mutation rate of the CRISPR-Cas9 vector in the crops, such as the wheat after transformation and difficulty in obtaining of the CRISPR-Cas9 transgenic mutant strain are solved; the transformation time is shortened; the transformation cost is reduced; a better foundation is laid for popularization and application of a CRISPR-Cas9 gene editing system in the crop.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a wheat genome editing vector based on a CRISPR-Cas9 system and an application thereof. Background technique [0002] Since the end of the 1980s, genetic modification technology based on artificial endonuclease began to develop, and currently mainly includes: the first generation of artificial endonuclease zinc finger nuclease (ZFN) technology, the second generation of artificial endonuclease transcription Activator-like effector nuclease (TALEN) technology, third-generation artificial endonuclease CRISPR-Cas9 nuclease technology. ZFN technology has poor specific recognition ability and is prone to off-target phenomena, causing other target gene mutations and chromosomal aberrations. In addition, the design screening of ZFNs is time-consuming and costly, thus limiting their wider application. Compared with ZFN technology, TALEN has less off-target probabilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22C12N15/113
CPCC12N9/22C12N15/113C12N15/8213C12N2310/12
Inventor 李健王峥马力耕邓兴旺
Owner BEIJING NEXT GENERATION HYBRID WHEAT BIOTECHNOLOGY CO LTD
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