Gene editing vector and gene editing method and application thereof

A gene editing and carrier technology, applied in the field of genetic engineering, can solve the problems of host limitations, limited host range, and insufficient host range, etc., and achieve high editing efficiency

Pending Publication Date: 2021-12-10
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the host range of TRV is relatively limited and cannot infect important crops such as barley, wheat, and corn
[0013] In summary, most of the current gene editing systems tha...

Method used

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  • Gene editing vector and gene editing method and application thereof
  • Gene editing vector and gene editing method and application thereof
  • Gene editing vector and gene editing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] This example constructs a CRISPR / Cas9 gene editing system using soybean mosaic virus as a carrier, the system is as follows figure 1 As shown, the build process is as follows:

[0068] 1. Select soybean mosaic virus SC7 isolate (Genbank Accession#: MH919385) as raw material;

[0069] 2. Codon-optimized nucleic acid sequence encoding capsid protein of soybean mosaic virus SC7 isolate. Using the online website (https: / / www.vectorbuilder.cn / tool / codon-optimization.html), one-click optimization of species selection common tobacco, the optimized nucleic acid sequence is obtained, as shown in SEQ ID NO.1, the optimized The nucleic acid sequence of soybean mosaic virus capsid protein was directly synthesized to replace the original CP sequence in the reading frame. (Note: The role of codon optimization is to avoid coincidence with the newly inserted CP sequence after the stop codon, resulting in virus-mediated homologous recombination, so that the gRNA in the midd...

Embodiment 2

[0096] This embodiment constructs the CRISPR / Cas9 gene editing system with the potato virus Y as the carrier, the system is as follows Figure 12 As shown, the build process is as follows:

[0097] 1. Select Potato virus Y ZT5 isolate (Genbank Accession#: MF960848) as raw material;

[0098] 2. Codon optimization of the nucleic acid sequence encoding the capsid protein of the potato virus Y ZT5 isolate. Using the online website (https: / / www.vectorbuilder.cn / tool / codon-optimization.html), one-click optimization of species selection common tobacco, the optimized nucleic acid sequence (SEQ ID NO.49), the optimized potato Y Direct gene synthesis of viral capsid protein nucleic acid sequence.

[0099] 3. Divide Potato virus Y into 5 segments for PCR amplification, and at the same time, hammerhead ribozyme, gRNA (editing PDS), and insert sequences are integrated into the infectious clone of Potato virus Y by PCR. The specific cloning steps are as follows :

[0100] (1) ...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a gene editing vector and a gene editing method and application thereof. The gene editing vector comprises a sequence of potyvirus from a 5'-UTR region to a capsid protein region, gRNA, an insertion sequence and a 3'-UTR region which are arranged in sequence, and the insertion sequence comprises a nucleic acid sequence for coding the virus capsid protein of the potyvirus. According to the gene editing vector, a gene editing system is constructed through the potyvirus, so that a variety of crops, such as soybeans and potatoes, which are less involved in the prior art can be dip-dyed, and the object and range of gene editing are expanded. Meanwhile, the technical problem that the gRNA is difficult to generate in a conventional mode due to the fact that the virus of the potyvirus cannot generate subgenome RNA is solved, and the method has important significance in the technical field of genetic engineering.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a gene editing vector and its gene editing method and application. Background technique [0002] In many bacteria and most archaea, there is a defense system that can be used to resist foreign DNA (such as phage), called CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated Cas9 endonuclease) system (Bhaya et al ., 2011). The CRISPR / Cas9 system consists of two basic components, one is the endonuclease Cas9 protein, and the other is gRNA (guide RNA). Under certain conditions, the gRNA can form a complex with the Cas9 protein, and through complementary base pairing, the gRNA can guide the Cas9 protein to the target DNA, and then the Cas9 protein can bind and cut the target double-stranded DNA, thereby causing the target DNA double-strand break ( double strand break). Intracellular DNA repair mechanisms repair damaged double-stranded...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/55
CPCC12N15/8203C12N9/22C12N2770/34043
Inventor 许凯张望智海剑
Owner NANJING NORMAL UNIVERSITY
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