CRISPR/Cas9 system and method for efficiently producing mutant plants without carrying transgenic elements in plants

A transgenic and plant technology, which is applied in the field of efficiently obtaining edited mutants without transgenic elements, and efficiently obtaining mutants that have undergone multiple editing and without transgenic elements, and can solve the problem of not discussing editing efficiency, editing element removal effect, No clear use, no research on the effects of editing on T2 plants, etc.

Active Publication Date: 2020-02-04
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It mentions the use of CaMV 35S promoter-driven Cas9-2A-GFP expression cassette in tobacco, but it is not clear what source of 2A peptide is used
Since this article focuses on the combined use of GFP and FACS sorting, it is limited to the

Method used

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  • CRISPR/Cas9 system and method for efficiently producing mutant plants without carrying transgenic elements in plants
  • CRISPR/Cas9 system and method for efficiently producing mutant plants without carrying transgenic elements in plants
  • CRISPR/Cas9 system and method for efficiently producing mutant plants without carrying transgenic elements in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1. Potency of 2A peptides from different sources

[0066] In order to obtain a CRISPR system capable of visually measuring the abundance of functional Cas9 in plants, the present inventors explored a variety of plant-applicable self-cleaving peptides for linking Cas9 and reporter proteins. Picornavirus-derived 2A peptides were shown to exhibit self-cleavage properties in more than 100 animal cells and tissues (Kim et al., PLoS One 6, e18556, 2011), to test whether 2A peptides could also function in plants To obtain a similar effect, and which 2A peptide has the best effect in plants, five different 2A peptides were tested as follows: Two 2A peptides derived from foot-and-mouth disease virus (F2A-1 and F2A-2, encoding nucleotides) The sequences are respectively SEQ ID NO: 2 and SEQ ID NO: 4), derived from the 2A peptide (E2A, the coding nucleotide sequence is SEQ ID NO: 3) of the corona virus, derived from the East Asia virus (Thosea asignavirus) The 2A peptid...

Embodiment 2

[0095] Example 2. Efficacy of different promoters

[0096] The promoter is one of the key factors in the CRISPR system that can affect the editing efficiency. There are some previous studies showing that the Cas9 system regulated by the 35S promoter is inefficient and that the induced somatic mutations cannot be inherited.

[0097] In order to select an efficient promoter to drive Cas9-P2A-GFP and to test whether GFP intensity correlates with gene editing efficiency, the Arabidopsis BRI1 (Brassinosteroid-Insensitive 1) gene was selected as the target gene to study different promoters, including dual CaMV 35S promoter and the Arabidopsis ubiquitin 10 (UBQ10) gene promoter, and tested different vectors, including pJim19 and pCAMBIA1300.

[0098] The BRI1 gene encodes a receptor protein with kinase activity that regulates the signaling cascade of growth and development by binding to brassinosteroid. If the BRI1 gene is edited so that it does not function properly, the edited ...

Embodiment 3

[0107] Example 3. Multiplex Editing Using Isocaudal Enzymes

[0108] In order to explore whether the system of the present invention is suitable for multiplex gene editing, the construct of the present invention was combined with an assembly method based on homocaudal restriction enzymes. Since the experiment in this example was performed before the experiment in Example 2, the 35S promoter was still used.

[0109] To generate expression cassettes for multiple sgRNAs, the pAtU6-26-SK vector (Feng et al., 2013, supra) was modified by deleting the Spe I site downstream of the sgRNA scaffold and adding a Nhe I site between Xho I and Sal I point to modify, get pAtU6-26-M ( Figure 5 ).

[0110] Multiple sgRNAs were inserted into the BbsI site of pAtU6-26-M. Assemble multiple sgRNA expression cassettes using Sal I and a pair of homologous restriction enzymes Spe I / Nhe I, such as Figure 1D shown.

[0111] To generate the final genome editing plasmid, the multiple sgRNA exp...

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Abstract

The invention relates to a construct for CRISPR/Cas9 gene editing in plants so as to efficiently produce genetically edited mutant plants without carrying transgenic elements. The invention also relates to a system and a method for carrying out CRISPR/Cas9 gene editing of plants by using the construct.

Description

technical field [0001] The invention belongs to the field of plant gene editing. Specifically, the present invention relates to a self-cleaving peptide-based construct for CRISPR / Cas9 gene editing in plants, and a system and method for efficiently obtaining edited mutants that do not carry transgenic elements using the construct . The present invention further provides a construct for CRISPR / Cas9 multiple gene editing in plants, and a system and method for efficiently obtaining multiple edited mutants without transgenic elements using the construct. Background technique [0002] The clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats; CRISPR) / CRISPR-associated (Cas) genome editing system derived from the adaptive immune system of type II prokaryotes is a precise gene editing system. It has been widely used in the research of functional genomes of animals and plants. A conventional CRISPR / Cas9 system contains...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/113C12N15/82A01H5/00
CPCC12N9/22C12N15/113C12N15/8218C12N2310/20
Inventor 陈浩东汪加军
Owner PEKING UNIV
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