Cell enrichment technology with C.T base substitution using inactive screening agent resistance gene as reporting system and application of technology

A technology of resistance gene and screening agent, applied in genetic engineering, recombinant DNA technology, introduction of foreign genetic material using vectors, etc.

Active Publication Date: 2019-12-31
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • 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

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  • Cell enrichment technology with C.T base substitution using inactive screening agent resistance gene as reporting system and application of technology
  • Cell enrichment technology with C.T base substitution using inactive screening agent resistance gene as reporting system and application of technology
  • Cell enrichment technology with C.T base substitution using inactive screening agent resistance gene as reporting system and application of technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Example 1, Establishment of Cell Enrichment Technology for C T Base Substitution

[0205] 1. Establishment of cell enrichment technology carrier for C T base substitution

[0206] 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.

[0207] 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 -ATG Schematic diagram of the structure of the / sgRNA-GT vector figure 2 shown.

[0208] The cell-free enrichment technology vector contains the complete selection agent resistance gene. The cell enrichmen...

Embodiment 2

[0214] Example 2, Construction of Cas9n&PmCDA1&UGI-mediated cell enrichment technology vector and its application in rice genome editing

[0215] 1. Construction of recombinant expression vector

[0216] 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.

[0217] 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

[0250] Example 3. Construction of HypaCas9n&PmCDA1&UGI-mediated cell enrichment technology vector and its application in genome editing of rice T0 seedlings

[0251] 1. Construction of recombinant expression vector

[0252] 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...

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Abstract

The invention discloses a cell enrichment technology with C.T base substitution using an inactive screening agent resistance gene as a reporting system and application of the technology. A vector of the cell enrichment technology comprises the following reagents: sgRNA, a C.T base substitution system and a screening agent resistance gene with loss of function. The sgRNA is composed of tRNA-sgRNA targeting a target sequence of an object gene and tRNA-sgRNA targeting a target sequence of the screening agent resistance gene with loss of function. The function of the screening agent resistance gene with loss of function can be recovered by performing C.T base substitution on the target sequence of the screening agent resistance gene with loss of function, by the C.T base substitution system, under the guide of tRNA-sgRNA targeting the target sequence of the screening agent resistance gene with loss of function. According to the present invention, cell enrichment with C.T base substitutionat the cell level is realized, and the efficiency of C.T base substitution is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a C·T base replacement cell enrichment technology using an inactivated screening agent resistance gene as a reporter system and its application. 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 ap...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/66C12N15/82C12N9/22
CPCC12N9/22C12N15/65C12N15/66C12N15/8209C12N15/8218
Inventor 徐雯杨进孝张成伟赵思冯峰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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