Application of difference agency technique to C. T base substitution cell enrichment

A base, biological cell technology, applied in the application field of differential surrogate technology in the enrichment of C·T base replacement cells

Active Publication Date: 2019-12-13
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
View PDF9 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there are very limited studies on the enrichment of C T base replacement cells in plants by reporter gene-mediated cell enrichment technology, and there is no use of screening markers used in the transformation process to achieve C T base substitution at the cellular level. Enrichment of replacement cells to improve C T base replacement efficiency report

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 difference agency technique to C. T base substitution cell enrichment
  • Application of difference agency technique to C. T base substitution cell enrichment
  • Application of difference agency technique to C. T base substitution cell enrichment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0208] Example 1, Establishment of Cell Enrichment Technology for C T Base Substitution

[0209] 1. Establishment of cell enrichment technology carrier for C T base substitution

[0210] The common technology (non-cell enrichment technology) carrier of Cas9 nuclease, cytosine deaminase and UGI-mediated C T base replacement was named sgRNA-GT. Taking the Cas9 nuclease as SpCas9n and the cytosine deaminase as PmCDA1 as an example: the structure diagram of the sgRNA-GT vector is as follows figure 1 shown.

[0211] The cell enrichment technology carrier of Cas9 nuclease, cytosine deaminase and UGI-mediated C T base replacement was named sgRNA -ATG -Hyg -ATG / sgRNA-GT. Take the Cas9 nuclease as SpCas9n and the cytosine deaminase as PmCDA1 as an example: sgRNA -ATG -Hyg -ATGSchematic diagram of the structure of the / sgRNA-GT vector figure 2 shown.

[0212] The cell-free enrichment technology vector contains the complete selection agent resistance gene. The cell enrichment...

Embodiment 2

[0218] Example 2, Construction of Cas9n&PmCDA1&UGI-mediated cell enrichment technology vector and its application in rice genome editing

[0219] 1. Construction of recombinant expression vector

[0220] The recombinant expression vector in this example is the non-cell enrichment technology carrier sgRNA-GT of Cas9n&PmCDA1&UGI (PCBE) mediated C T base substitution and the cell enrichment of Cas9n&PmCDA1&UGI (PCBE) mediated C T base substitution Technical carrier sgRNA -ATG -Hyg -ATG / sgRNA-GT. Each vector is a circular plasmid. The structural diagrams of the components of the two recombinant expression vectors are as follows: figure 1 and figure 2 shown.

[0221] According to the different target sequences contained, each recombinant expression vector is divided into two types, and there are four recombinant expression vectors as follows: sgRNA -ATG -Hyg -ATG / sgRNA-GT-1 recombinant expression vector, sgRNA -ATG -Hyg -ATG / sgRNA-GT-2 recombinant expression vector, ...

Embodiment 3

[0254] Example 3. Construction of HypaCas9n&PmCDA1&UGI-mediated cell enrichment technology vector and its application in genome editing of rice T0 seedlings

[0255] 1. Construction of recombinant expression vector

[0256] The recombinant expression vector in this example is a non-cell enrichment technology carrier (named HypaCas9n-sgRNA-GT) mediated by HypaCas9n&PmCDA1&UGI (HypaCas9-PCBE) mediated C T base replacement and HypaCas9n&PmCDA1&UGI (HypaCas9-PCBE) mediated Cell enrichment technology carrier (named sgRNA -ATG -Hyg -ATG / HypaCas9n-sgRNA-GT). Each vector is a circular plasmid. The structural diagrams of the components of the two recombinant expression vectors are as follows: Figure 7 shown. The main structure of the carrier is similar to that of Cas9n&PmCDA1&UGI-mediated non-cell enrichment technology carrier and cell enrichment technology carrier, the only difference is that HypaCas9n is used instead of SpCas9n. The working principle of HypaCas9n&PmCDA1&UGI-m...

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 an application of a difference agency technique to C. T base substitution cell enrichment. A difference agency technique carrier disclosed by the invention comprises the following reagents: sgRNA, a C. T base substitution system and a functional incapacitation screening agent resistant gene, wherein the sgRNA consists of esgRNA targeting a target point sequence of an objective gene and sgRNA targeting a target point sequence of the functional incapacitation screening agent resistant gene; the C.T base substitution system can perform C. T base substitution on the targetpoint sequence of the functional incapacitation screening agent resistant gene under guidance of the sgRNA for targeting the target point sequence of the functional incapacitation screening agent resistant gene so that the functions of the functional incapacitation screening agent resistant gene are recovered. According to the application disclosed by the invention, C.T base substitution cell enrichment on cell level can be realized, and the C.T base substitution efficiency is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of differential agent technology in C·T base substitution cell enrichment. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base substitution ...

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): C12N15/65C12N15/82
CPCC12N15/65C12N15/8209
Inventor 杨进孝徐雯杨永星康桂婷武莹李璐
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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