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Fluorescence labeled CRISPR/SpCas9 system mediated gene replacement system and application thereof in plants

A spcas9-t2a-gfp, fusion gene technology, applied in the field of target gene homologous recombination, can solve the problems of increasing off-target risk, heavy workload, easy pollution and the like

Inactive Publication Date: 2021-04-20
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because plants carrying CRISPR / SpCas9 elements are transgenic plants, it not only increases the risk of off-target, but also is not conducive to subsequent functional analysis
The offspring can obtain non-transgenic plants through natural separation or crossing with wild type, but the identification process has problems such as heavy workload and easy pollution

Method used

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  • Fluorescence labeled CRISPR/SpCas9 system mediated gene replacement system and application thereof in plants
  • Fluorescence labeled CRISPR/SpCas9 system mediated gene replacement system and application thereof in plants
  • Fluorescence labeled CRISPR/SpCas9 system mediated gene replacement system and application thereof in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Construction of vector pCXUN-Ubi-NLS-SpCas9-T2A-GFP-Nos-OsU3-gRNA-Actin-RDR-S189A / S189L-Nos

[0058] In this example, a vector pCXUN-Ubi-NLS-SpCas9-T2A-GFP-Nos-OsU3-gRNA-Actin-RDR-S189A / S189L-Nos was constructed, which can realize the homologous recombination of the target gene (RLI1 gene) in rice. The specific method is as follows:

[0059] 1. Construction of expression vector

[0060] 1. Construction of plasmid pCXUN-Ubi-NLS-SpCas9-T2A-GFP-Nos

[0061] (1) The plasmid pCXUN-SpCas9 was double digested with restriction endonucleases StuI and AflII to obtain a vector backbone 1 of about 15350 bp.

[0062] (2) Using the chemically synthesized SEQ ID No.2 (that is, the T2A-GFP fragment with sequences homologous to the two ends of the backbone 1) as a template, a primer pair consisting of primer StuI-F and primer AflII-R was used for PCR amplification, the 5' and 3' end sequences of the PCR product are completely consistent with the two end sequences of the ve...

Embodiment 2

[0080] Example 2. Using CRISPR / SpCas9 system to realize homologous recombination of RLI1 gene (target gene) in rice

[0081] 1. Obtaining genetically modified rice

[0082] 1. Select the plump Zhonghua 11 rice seeds, peel off the seed coat, sterilize and wash, put them evenly in the sterilized NB solid medium containing 3.5mg / L2, 4-D, and cultivate in the dark at 28°C for 30-40d to induce callus production.

[0083] 2. After the callus obtained in step 1 was hyperosmotically treated in NB medium containing 0.3M mannitol and 0.3M sorbitol for 4-6h, the pCXUN-Ubi-NLS-SpCas9-T2A-GFP- The Nos-OsU3-gRNA-Actin-RDR-S189A / S189L-Nos plasmid bombarded rice callus by gene gun, using 0.6μm gold powder, bombardment pressure of 900psi for bombardment, after bombardment in NB containing 0.3M mannitol and 0.3M sorbitol After being cultured in the culture medium for 16 hours, they were transferred to N6 selection medium (NB solid medium containing 0.5 mg / L 2,4-D and 50 mg / L hygromycin), and ...

Embodiment 3

[0094] Embodiment 3 detects the separation situation of T1 seed offspring

[0095] Collect wild type Zhonghua 11 rice (ZH11), T prepared in Example 2 1 Generation transgenic plant (#2-1) and the T prepared in embodiment 2 1 The seeds of transgenic plants (#1-8) of the first generation, after the seeds germinated, the fluorescence was detected by a fluorescence microscope. The genomic DNA of the above seeds was extracted, and the genomic DNA was used as a template to carry out PCR amplification using a primer pair composed of primer RLI1 TestF and primer RLI1 TestR. The schematic diagram of PCR amplification is shown in figure 1 B in

[0096] Figure 5 for part T 1 Generation seed fluorescence observation results, where, BF: Bright-field: bright field, Flu: Fluorescence, fluorescence. Such as Figure 5 As shown, seeds harvested from T1 plants containing the GFP expression cassette were divided into two groups: one group showed strong green fluorescence and the other gro...

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Abstract

The invention discloses a recombinant vector, a DNA molecule, application and a method for carrying out homologous recombination on a target gene by taking an RNA transcript as a template in a plant. The recombinant vector comprises an expression cassette A, an expression cassette B and an expression cassette C; the expression cassette A comprises a promoter A, SpCas9-T2A-GFP and a terminator; the expression cassette B comprises a promoter B and an encoding gene of gRNA, and a target fragment targeted by the gRNA is a DNA fragment to be replaced in a plant genome; the expression cassette C comprises a promoter C, an encoding gene of nuclease A, a template section and an encoding gene of nuclease B, and the template section comprises an upstream homologous arm of the target fragment, a donor fragment and a downstream homologous arm of the target fragment; and the donor fragment has a difference from the target fragment as follows: the donor fragment contains different nucleotides expected to be introduced into the target fragment. According to the invention, the recombinant vector utilizes a CRISPR / SpCas9 system, RNA serves as a homologous recombination repair template, replacement of a target gene is achieved in rice, and the application range of TT-HDR to the CRISPR / SpCas9 system is expanded.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for homologous recombination of target genes using RNA transcripts as templates in recombinant vectors, DNA molecules, applications and plants. Background technique [0002] Since the CRISPR / SpCas9 system was first reported to edit plant genomes in August 2013, a large number of related studies have been carried out. In these studies, plants such as Arabidopsis thaliana, tobacco, rice, and wheat were used as test materials, and the optimized SpCas9 gene and gRNA were transferred into plants through transgenic technology. The 20bp target sequence of PAM (NGG) is edited to cause double-strand breaks (double-strand breaks, DSBs) in the genomic DNA sequence of the target site, and then through non-homologous end joining (NHEJ) or Homologous recombination-mediated repair (homology-directed repair, HDR) introduces mutations in two ways, thus realizing the site-directed editing of...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N9/22C12N15/65
Inventor 夏兰琴李慧园闫磊
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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