Application of CRISPR/nCas9 mediated site-directed base substitution in plant

A plant and gene technology, applied in the application field of fixed-point base replacement in plants, can solve the problem that agronomic traits cannot be improved quickly

Active Publication Date: 2017-08-15
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
View PDF1 Cites 59 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of agronomic traits in crops are caused by mutations of single bases. After the introduction of DSB by traditional CRISPR/Cas9 technology, HDR always occurs at a rather low frequency compared with the random process of non-homologous ...

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Application of CRISPR/nCas9 mediated site-directed base substitution in plant
  • Application of CRISPR/nCas9 mediated site-directed base substitution in plant
  • Application of CRISPR/nCas9 mediated site-directed base substitution in plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Application of a CRISPR / nCas9-mediated site-directed base replacement in plants

[0082] 1. Construction of expression vector

[0083] 1. Construction of pCXUN-BE3 vector

[0084] (1) Digest the pCXUN-Cas9 vector with restriction endonuclease BamHI to obtain a linearized vector;

[0085] (2) Perform PCR amplification with BE-F / R as primers and pCMV-BE3 vector as template to obtain a PCR product, the 5' and 3' end sequences of the PCR product are completely consistent with the sequences at both ends of the linearized vector ;

[0086] (3) The linearized vector obtained in step (1) and the PCR product obtained in step (2) were connected by homologous recombination using the pEASY-Uni Seamless Cloning and Assembly Kit of Quanshijin Company to obtain the vector pCXUN-BE3( figure 1 ), as can be seen from the figure: the pCXUN-BE3 vector includes the expression cassette A, which in turn includes the maize Ubiquitin promoter, the gene encoding deaminase (APOBEC1)...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses application of CRISPR/nCas9 mediated site-directed base substitution in a plant. The invention provides a plant genome site-directed edition system. The system comprises a BE3 plant expression carrier (expressing a fusion protein composed of nCas9(D10A), deaminase and a uracil DNA glycosylase inhibitory protein), and rice OsPDS and OsSBEIIb are taken as target genes for verifying the system. Results show that an expected site-directed mutant plant is respectively obtained in the three selected target spots, accurate site mutation of a base is realized in rice, and the highest efficiency reaches about 20%, so that a feasible and effective base substitution method is provided for crop breeding, the method has strong application potential in the aspect of agricultural breeding, and a foundation is provided for rapidly improving important agronomic traits of crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an application of CRISPR / nCas9-mediated site-directed base replacement in plants. Background technique [0002] CRISPR / Cas9-mediated genome editing technology has become one of the most powerful tools in molecular biology. Found in bacteria for the first time, it consists of two parts, sgRNA and Cas9 (Jinek et al., 2012). CRISPR / Cas9 causes double-strand breaks (double-strand breaks, DSBs) at the target site through its own endonuclease activity, and then through non-homologous end joining (NHEJ) or homologous recombination Homology-directed repair (HDR) introduces mutations in two ways. Most of the mutations induced by the NHEJ pathway are insertions or deletions of nucleotides, resulting in frameshift mutations, while in HDR, fragment insertions or nucleotide corrections are mediated by homologous donor DNA (Jinek et al., 2012). The recognition of target sites by the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8201C12N15/8222C12N2810/10
Inventor 夏兰琴孙永伟赵云德李晶莹杜晋鲁
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap