CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system

An allele and fragment technology, applied in the field of allele replacement, can solve the problems of time-consuming and laborious, low HDR frequency, etc., and achieve the effect of great application potential and application prospect.

Inactive Publication Date: 2018-03-13
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
View PDF4 Cites 32 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The introduction of these genes or excellent traits through conventional hybridization and backcrossing transfer methods requires years of multi-generation material selection, which is time-consuming and laborious
Although CRISPR / Cas9, as a new targeted gene modification technology, has shown broad development potential and application prospects, its application in crop improvement still has certain limitations. HDR occurs particularly infrequently compared to the random repair process of end junctions

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
  • CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system
  • CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system
  • CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Preparation of recombinant plasmid pCXUN-cas9-gRNA1-gRNA2-arm donor and free donor fragment

[0051] The free donor fragment is shown in sequence 4 of the sequence listing. The schematic diagram of the structure of the free donor fragment is shown in figure 1 shown. In sequence 4 of the sequence listing, the 4th-26th nucleotide is target 1 (Target1) [the 4th-23rd nucleotide is the target sequence of sgRNA1, and the 24th-26th nucleotide is CGG], the 30th -The 129th nucleotide is the upstream homology arm (Left Arm), the 130th-152nd nucleotide position is mutated target 1 (Mutated target1) [the 130th-149th nucleotide is the target sequence of sgRNA1, the 150th -The 152nd nucleotide is CCG], the 155th-157th nucleotide is ATG (coding methionine) [that is, causes the T327M mutation in the protein], the 296th-301st nucleotide is GAACTC (Mutated SacⅠ )[is about to mutate the original restriction endonuclease SacⅠ "GAGCTC"], the 358th-380th nucleotide is the mutat...

Embodiment 2

[0054] Example 2, Application of recombinant plasmid pCXUN-cas9-gRNA1-gRNA2-arm donor and free donor fragments to introduce site-directed mutations in the NRT1.1B gene

[0055] 1. Obtaining genetically modified rice

[0056] 1. Take the plump 11 seeds of Zhonghua, peel off the seed coat, sterilize and wash in sequence, then place on the induction medium, and culture in the dark at 28°C for 40-50 days to induce the generation of callus.

[0057] Induction medium: solid NB medium containing 2mg / L 2,4-D.

[0058] 2. After completing step 1, take the callus and treat it on the hyperosmotic medium for 4-6 hours,

[0059] Hypertonic medium: solid NB medium containing 0.3M mannitol and 0.3M sorbitol.

[0060] 3. Mix the recombinant plasmid pCXUN-cas9-gRNA1-gRNA2-arm donor and the free donor fragment at a molar ratio of 1:20, and then bombard the callus that completed step 2 with a gene gun (using 0.6 μm gold powder, the bombardment pressure is 900psi).

[0061] 4. Take the callus...

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 a CRISPR-mediated rapid and effective crop site-specific gene fragment or allele replacement method and system. The system provided by the invention comprises a recombinant vector and a free donor fragment; the recombinant vector comprises a coding sequence of sgRNA1, an expression cassette of a Cas9 gene, a donor fragment and a coding sequence of sgRNA2; the donor fragmentcomprises a target of sgRNA1, a mutant segment and a target of sgRNA2; homologous recombination occurs between the mutant segment and a target segment; and the differences between the mutant segmentand the target segment are as follows: (1) a target nucleotide or fragment is replaced with a mutated nucleotide or fragment, (2) NGG in the target of sgRNA1 is mutated into non-NGG, and (3) the NGG in the target of sgRNA2 is mutated into non-NGG. The invention provides a feasible and effective method for replacing and integrating site-specific gene fragments or alleles for crop breeding improvement, and the method has a great application potential and a wide prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR-mediated fast and effective crop site-specific gene segment or allele replacement method and system. 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. 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. The recognition of target sites by the CRISPR / Cas9 s...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/89A01H5/00
CPCC12N15/82C12N15/895C12N2800/80C12N2810/10
Inventor 夏兰琴李晶莹赵云德孙永伟张佳慧
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