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CRISPR/RfxCas13d anti-plant RNA virus vector and construction method and application of CRISPR/RfxCas13d anti-plant RNA virus vector

A technology of RNA virus and construction method, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effects of precise targeting, efficient cutting of RNA, and wide recognition

Inactive Publication Date: 2020-08-18
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no effective means to prevent and control the infection of TuMV

Method used

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  • CRISPR/RfxCas13d anti-plant RNA virus vector and construction method and application of CRISPR/RfxCas13d anti-plant RNA virus vector
  • CRISPR/RfxCas13d anti-plant RNA virus vector and construction method and application of CRISPR/RfxCas13d anti-plant RNA virus vector
  • CRISPR/RfxCas13d anti-plant RNA virus vector and construction method and application of CRISPR/RfxCas13d anti-plant RNA virus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of CRISPR / RfxCas13d anti-plant RNA virus vector

[0028] The technical route for constructing the carrier is as follows:

[0029] 1. Construction of pCambia1300-RfxCas13d recombinant plasmid

[0030] A 3003bp RfxCas13d gene fragment was artificially synthesized and cloned into pUC57, named pUC57:RfxCas13d (Sangon Bioengineering (Shanghai) Co., Ltd.). Plasmids pUC57: RfxCas13d and pCambia1300-spCas9 (purchased from Hangzhou Baige Biotechnology Co., Ltd.) were double digested with BamHI and NcoI, and the 3003bp RfxCas13d gene fragment and the pCambia1300 vector backbone of about 10kb were recovered respectively. T4 DNA ligase (Thermo Cat #EL0011) connected the 3003bp RfxCas13d fragment to the pCambia1300 vector backbone, and the plasmid was named pCambia-RfxCas13d. Store the plasmid after sequencing, and store the Agrobacterium after successful verification.

[0031] Such as figure 1 Shown, the composition of plasmid pCambia1300-RfxCas13d is as ...

Embodiment 2

[0037] Example 2: The use of pCambia1300-RfxCas13d-crGFP can inhibit the infection of TuMV-GFP

[0038] 1. Agrobacterium plant infection and inoculation

[0039] (1) Activate Agrobacterium, inoculate the Agrobacterium liquid containing pCambia1300-RfxCas13d (Cas13d for short), pCambia1300-RfxCas13d-crGFP (Cas13d-crGFP for short), and TuMV-GFP plasmid to OD 600 At about 1.5, centrifuge at 4,000rpm for 12min to collect the bacteria, resuspend the bacteria in a double distilled water solution (infiltration buffer) containing a final concentration of 10mM magnesium chloride, 10mM MES (pH5.6) and 100mM acetosyringone, and adjust the bacteria solution concentration to make it OD 600 It is about 1.0 and placed at room temperature for 3 hours. Infectious clones of Cas13d-crGFP and TuMV-GFP Agrobacterium were injected simultaneously, or invasive clones of Cas13d-crGFP and TuMV-GFP Agrobacterium ( image 3 is the vector diagram of TuMV-GFP), the concentration of bacterial solution sh...

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Abstract

The invention discloses a CRISPR / RfxCas13d anti-plant RNA virus vector and a construction method and application thereof. The vector comprises a pCambia 1300-RfxCas13d recombinant vector, the recombinant vector is composed of a CaMV 35S promoter, a hygromycin gene, a CaMV poly(A) signal terminator, a pVS1 RepA, a pVS1 replication starting point, a CaMV 35S promoter, an RfxCas13d gene and an NOS terminator, the sequence of the RfxCas13d gene is as shown in SEQ ID No. 1, and the sequence of the NOS terminator is as shown in SEQ ID No. 2. Turnip mosaic virus (TuMV-GFP) infectious clone with a GFPfluorescent label is used for indicating RNA viruses to naturally invade plants, and RNA can be directionally cut at a specific position through combination of RfxCas13d protein and sgRNA. The vectorconstructed by the invention can effectively inhibit infection of TuMV, provides a new strategy for prevention and control of plant virus diseases, and has important production significance.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-efficiency site-directed editing CRISPR / RfxCas13d anti-plant virus vector and its construction method and application. Background technique [0002] Genome fixed-site editing technology is an effective and high-quality technical means, which can be used in plant functional genome research and crop molecular genetic breeding. Genome editing technology mainly uses the following three artificial endonucleases: zinc finger nucleases ZFNs, transcription activator-like effector nucleases TALENs, and RNA-guided endonucleases based on the CRISPR / Cas system. The process of gene editing against DNA is to generate double-strand DNA breaks (DSBs) at genomic target sites, and DSBs can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HDR). Endonucleases in the RNA-guided endonuclease system of the CRISPR-Cas system, such as SpCas9 and LbCpf1, have been shown t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/55
CPCC12N15/8218C12N15/8283C12N15/66C12N9/22
Inventor 李方方曹永森周雪平
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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