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Construction method and application of high-efficiency soybean CRISPR/Cas9 system

A construction method and high-efficiency technology, applied in the field of genetic engineering, can solve problems such as editing efficiency needs to be further optimized, stable genetic transformation system to verify editing efficiency, etc.

Inactive Publication Date: 2018-09-28
NANCHANG UNIV
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Problems solved by technology

According to the reported data, in 2015, SpCas9 has been applied to soybean cotyledon transient transformation and stable genetic transformation system, but the editing efficiency needs to be further optimized
In 2016, Cpf1 was also used in soybean protoplasts, but its editing efficiency was not verified in a stable genetic transformation system

Method used

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  • Construction method and application of high-efficiency soybean CRISPR/Cas9 system
  • Construction method and application of high-efficiency soybean CRISPR/Cas9 system
  • Construction method and application of high-efficiency soybean CRISPR/Cas9 system

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Embodiment 1

[0033] The sources of reagents and materials used in this embodiment:

[0034] 1) The pCAMBIA1300 vector is owned by the experiment;

[0035] 2) Primer synthesis and sequencing are entrusted to Beijing Qingke Xinye Biotechnology Co., Ltd.;

[0036] 3) The soybean variety is YC03-3;

[0037] 4) The reagents used to construct the vector are Vazyme ClonExpress Seamless Cloning Kit, NEB T4ligase, TIANAprep Mini Plasmid Kit II Plasmid Extraction Kit, TaKaRa MiniBest DNA FragmentPurification Kit, NEB BsaI enzyme, etc.

[0038] 1. Construction of Soybean CRISPR / Cas9 Editing Vector

[0039] 1. Pick a single colony of the pCAMBIA1300 plasmid, inoculate it in 50ml of liquid LB medium containing kana antibiotics, place it on a shaker at 37°C, and culture it at 220rpm / r overnight;

[0040] 2. Collect the bacteria by centrifugation, and extract the plasmid with TIANAprep Mini Plasmid Kit II plasmid extraction kit;

[0041]3. Select a suitable enzyme cutting site, digest the pCAMBIA1300...

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Abstract

The invention discloses a construction method and an application of the high-efficiency soybean CRISPR / Cas9 system. Staphylococus aureus Cas9 protein undergoes split expression; and in order to improve editing efficiency and broad spectrum, a soybean SaCas9 editing system is constructed. According to the construction method provided by the invention, a low-efficiency editing system is transformedinto the high-efficiency editing system, so that the application scope of editing soybean genome is widened, and meanwhile, a new thought is provided for solving a problem on crop genome editing efficiency in the future.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application of a high-efficiency soybean CRISPR / Cas9 system. Background technique [0002] CRISPR / Cas9 is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The CRISPR / Cas9 system confers immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and using corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences. [0003] This discovery was purely accidental, and at first it did not attract too much attention, including the discoverer himself. Even this characteristic sequence did not have a name. Until 2002, many prokaryotes (eubacteria and archaea) were discovered through computer operations. After having a non-repeated sequence separated by a palindromic repeat sequence of 21-37bp, th...

Claims

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Application Information

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IPC IPC(8): C12N15/90A01H5/00A01H6/54
CPCC12N15/90C12N2800/80C12N2810/10
Inventor 王东唐雅君贺热情朱友林
Owner NANCHANG UNIV
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