CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode

A technology for encoding genes and plants, applied in angiosperms/flowering plants, plant products, genetic engineering, etc., can solve the problem of low editing efficiency

Active Publication Date: 2015-12-23
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of plants, this technology has also been applied to plants such as Arabidopsis thaliana, rice, corn, tobacco, and tomato, but the editing efficiency of the existing CRISPR / Cas9 system is relatively low
[0003] At present, the promoters used to drive the expression of Cas9 genes are mostly CMV35S promoter and Ubiquitin promoter, but existing studies have shown that the editing efficiency of Cas9 driven by both of them on the plant genome is low. It can be seen that choosing a suitable promoter to drive Expression of the Cas9 gene is especially important for improving its editing efficiency

Method used

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  • CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode
  • CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode
  • CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, construction of recombinant plasmid

[0061] 1. Construction of recombinant plasmid pYAO:Cas9

[0062] 1) Using the genomic DNA of wild-type Arabidopsis as a template, artificially synthesized pYAO-F: 5'-AA GTC GAC GATGGGAAATTCATTGAAAACCCT-3' (SalI restriction site is underlined) and pYAO-R: 5'-AA GTC GAC TCCTTTTCTTCTTCTCGTTGTTGT-3' (the underlined SalI restriction site) was used as a primer, and KOD-Plus-Neo was used for PCR amplification to obtain a double-stranded DNA molecule containing restriction endonuclease SalI at both the N-terminus and the C-terminus.

[0063] 2) After completing step 1), single-digest the double-stranded DNA molecule obtained in step 1) with restriction endonuclease SalI, and recover Fragment 1 of about 1024bp.

[0064] 3) The vector 35S-Cas9-SK was single-digested with the restriction endonuclease XhoI, and the vector backbone 1 of about 7493 bp was recovered.

[0065] 4) Ligate the fragment 1 with the vector backbone 1 t...

Embodiment 2

[0096] Example 2. Site-directed editing of Arabidopsis endogenous gene BRI1 by pYAO:Cas9 / AtU6-26-sgRNA system

[0097] 1. Design of target fragment BRI1-T1

[0098] Design the target fragment BRI1-T1, the target fragment BRI1-T1 is located on the target gene, and one strand in the double-stranded target fragment has the following structure: 5'-N X -NGG-3', N represents any one of A, G, C and T, X=20.

[0099] The nucleotide sequence of the target fragment BRI1-T1 is: 5'-TTGGGTCATAAC GATATC TC-3' (underlined is EcoRV enzyme recognition site).

[0100] 2. Construction of recombinant plasmid pYAO:hspCas9-BRI1-sgRNA

[0101] (1) Synthetic BRI1-T1F: 5'- ATTG TTGGGTCATAACGATATCTC-3' (the underlined part is sticky end) and BRI1-T1R: 5'- AAAC GAGATATCGTTATGACCCAA-3' (the underlined part is the cohesive end), BRI1-T1F and BRI1-T1R are all single-stranded DNA molecules.

[0102] (2) Mix BRI1-T1F and BRI1-T1R at a molar ratio of 1:1 and anneal (annealing procedure: 95°C, 5min, n...

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Abstract

The invention discloses a first expression cassette containing a promoter pYAO. In the expression cassette, the promoter pYAO promotes a coding gene expression of Cas9 nuclease; the promoter pYAO is shown in the following steps that (a1), the promoter pYAO is a DNA molecule at the 1-1012 position from the 5' tail end in a sequence table first sequence; (a2), the promoter pYAO is a DNA molecule which has 75% or more of identity with a nucleotide sequence which is defined by (a1) and has a promoter function; (a3), the promoter pYAO is a DNA molecule which hybridizes with the nucleotide sequence defined by (a1) or (a2) under strict conditions and has a promoter function. Experiments prove that YAO genes of high-expression genes at a plant gametophte or / and embryonic development early-stage are utilized for driving the expression of Cas9 genes, and the plant gene groups can be efficiently edited.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CRISPR / Cas9 system for efficiently editing plant genomes. Background technique [0002] Achieving efficient and targeted editing of plant genomes is of great significance for the study of plant gene functions. At present, gene modification technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN) and CRISPR / Cas9 have been widely used in scientific research, among which CRISPR / Cas9 technology is a gene that has only been developed in recent years. Retrofit technology. The CRISPR / Cas system is an acquired immune system found in most bacteria and all archaea to eliminate foreign plastids or phages and leave foreign gene fragments in their own genomes as "memory". Using the CRISPR / Cas9 system to edit biological genomes to cause different forms of deletions or insertions at target fragments has been successfully applied to human cell li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/113A01H5/00
CPCC12N15/8261Y02A40/146C12N15/8213C12N2310/20C12N15/113
Inventor 谢旗闫留华魏绍巍杨维才李红菊
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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