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Plant genome directional base editing skeleton vector and application thereof

A plant genome and base editing technology, which is applied in the field of plant genome directed base editing backbone vectors, can solve the problems of low editing efficiency and limited editing window, and achieve the goal of expanding the base editing window, good application prospects, and efficient directed editing Effect

Active Publication Date: 2019-12-24
UNIV OF ELECTRONICS SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing directional base editing tools generally have the problems of low editing efficiency and limited editing window, especially in the application practice of plant genome editing. Enhanced plant-directed base editing tool for base editing window, in order to effectively expand the active application of CRISPR-Cas system-based directed base editing technology in plant genome function research and breeding practice

Method used

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  • Plant genome directional base editing skeleton vector and application thereof
  • Plant genome directional base editing skeleton vector and application thereof
  • Plant genome directional base editing skeleton vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Construction of Plant STU nCas9-PmCDA1 Single Transcription Unit Directed Base Editing Backbone Vector

[0065] The present invention designs a STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier for plant genome engineering, and its core unit is pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold from 5' to 3' direction -HSP T. Among them: pZmUbi1 is the maize pZmUbi1 promoter (the basic STU nCas9-PmCDA1 single transcription unit-directed base-editing backbone carrier scheme can be used to realize the replacement of different PolII-type promoters through AscI and SbfI double enzyme digestion); nCas9 ORF is the suppurative chain Cocci (Streptococcus pyogenes) nuclease protein D10A mutant coding frame (including C-terminal NLS signal peptide); region, NLS signal peptide, UGI coding region, SGGS linker, NLS signal peptide); Poly A is the poly A region; sgRNA cloning scaffold (abbreviated as sgRNA CS) is the sgRNA clone and tr...

Embodiment 2

[0074] Example 2 High-throughput identification of rice endogenous gene cytosine-directed base editing efficiency based on STU nCas9-PmCDA1 system

[0075] 1. Rice endogenous gene sgRNA design and STU nCas9-PmCDA1 base editing recombinant expression vector construction

[0076] For rice OsCDC48 (LOC_Os03g05730) and OsROC5 (LOC_Os02g45250) coding genes, the 5'-NGG-3'PAM site was searched, and the 20bp sequence upstream of the PAM was selected to design sgRNA (Table 1).

[0077] Table 1 Design, synthesis and detection information of rice endogenous gene sgRNAcrRNA

[0078]

[0079] According to the designed sgRNA site nucleic acid sequence, artificially synthesize the corresponding forward and reverse oligonucleotide chains, the specific sequence is as follows (uppercase base sequence represents the designed site-specific guide sgRNA site; lowercase base sequence represents the site with Backbone vector complementary cohesive ends):

[0080] BE-OsCDC48-sgRNA01-F (Seq ID No....

Embodiment 3

[0095] Example 3 Based on the STU nCas9-PmCDA1 system, the creation and efficiency analysis of rice endogenous gene cytosine-directed base editing regenerated plants

[0096] 1. Agrobacterium transformation of rice endogenous gene STU nCas9-PmCDA1-sgRNA base editing recombinant expression vector

[0097]The STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors successfully constructed in Example 2 and detected in rice protoplasts for directional modification activity were passed The competent cells of Agrobacterium EHA105 were transformed by the heat shock method, spread on LB solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and cultured in the dark at 28°C for 2 days to obtain positive clones. Positive clones were activated in LB liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin for subsequent transformation.

[0098] 2. Agrobacterium-mediated rice callus transforma...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a plant genome directional base editing skeleton vector and an application thereof. The technical problemto be solved by the invention is to improve the directed base editing efficiency of a plant cell genome and expand a base editing window. The technical scheme for solving the technical problem is to provide the plant genome directional base editing skeleton vector. The skeleton vector is characterized in that a PolII type promoter drives transcription of a core unit consisting of two core regions,i.e., an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription expression unit. The single transcription unit oriented base editing skeleton vector can effectively realize simple, rapid and efficient oriented editing of converting cytosine bases (C) into thymine bases (T), and is a molecular tool for effectively realizing orientedediting of plant genome bases.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a plant genome-directed base editing backbone carrier and an application thereof. Background technique [0002] Targeted genome modification has always been a frontier and hot spot in biological research. Through precise targeted modification of specific regions of the genome: on the one hand, it can precisely mutate the target sequence, obtain mutation materials, and clearly identify the function of the target gene; on the other hand, it can Precise replacement or insertion of the target sequence minimizes the uncertainty of expression and inheritance caused by the random introduction of foreign genes. [0003] In 2012, researchers proved for the first time that CRISPR-Cas (Clustered regularly interspacedshort palindromic repeats-CRISPR associated protein) can achieve sequence-specific DNA double-strand shearing, and then the CRISPR-Cas9 system was used in cynomolgus monke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8213C12N2810/10
Inventor 张勇唐旭郑雪莲任秋蓉周建平邓科君
Owner UNIV OF ELECTRONICS SCI & TECH OF CHINA
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