Method for Rapidly Constructing Cas9 Binary Expression Vector Library of Paired sgRNA

A binary expression vector, phlw-grna-cas9-atu3b technology, applied in the field of rapid construction of binary vectors

Active Publication Date: 2021-09-03
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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Problems solved by technology

But so far there is no method for constructing a paired sgRNA Cas9 binary expression vector library for plants

Method used

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  • Method for Rapidly Constructing Cas9 Binary Expression Vector Library of Paired sgRNA
  • Method for Rapidly Constructing Cas9 Binary Expression Vector Library of Paired sgRNA
  • Method for Rapidly Constructing Cas9 Binary Expression Vector Library of Paired sgRNA

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Embodiment Construction

[0025] Below in conjunction with embodiment technical scheme of the present invention will be further described, but should not be interpreted as limitation of the present invention:

[0026] The source of biological material of the present invention:

[0027] 1. Vector pYLCRISPR / Cas9P-35S-N, pYLsgRNA-AtU3b: Ma, X., Zhang, Q., Zhu, Q., Liu, W., Chen, Y., Qiu, R. (2015) A Robust CRISPR / Cas9System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant, 8, 1274–1284, these two carriers were presented to the applicant by Professor Liu Yaoguang of South China Agricultural University;

[0028] 2. All primers are designed by the applicant of the present invention and commissioned by Shanghai Biological Biotechnology Co., Ltd. to synthesize;

[0029] 3. All PCR polymerases used were purchased from Beijing Quanshijin Co., Ltd.

[0030] 4. All restriction enzymes and T4 DNA ligase used were purchased from New England Biolabs.

[0031] This ex...

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Abstract

The invention discloses a method for rapidly constructing a Cas9 binary expression vector library paired with sgRNA, and relates to a method for rapidly constructing a binary vector in the field of genetic engineering. In this method, on the basis of constructing the CRISPR-Cas9 binary expression vector, the corresponding forward and reverse primers are designed according to the specific gene design target sequence, and the primer has two BsaI restriction sites; the target sequence and two BsaI sites are obtained by one-step PCR The target fragment, and then construct the above fragment into a binary expression vector with Cas9 gene by simple digestion and ligation to form a paired sgRNA Cas9 vector. The invention can efficiently establish random libraries and non-random libraries, which can be used for large-scale screening of gene functions in plants, and can be used for the construction and research of gene interaction networks between multiple genes; the experimental results prove that this method successfully and efficiently constructs Cas9 binary expression vector library of random paired sgRNAs for 14 targets (from 14 genes).

Description

technical field [0001] The invention relates to a method for rapidly constructing a binary vector in the field of genetic engineering, in particular to a method for rapidly constructing a Cas9 binary expression vector library paired with sgRNA. Background technique [0002] Based on the convenience and high efficiency of the CRISPR / Cas9 system, it has been successfully used for genome editing in eukaryotes and prokaryotes, especially in plants and animals. By further improving and optimizing the system, the system has been successfully used in gene transcription regulation, cell tracking, fluorescent labeling, epigenetic modification and single base mutation, etc. When this system is used for gene mutation, large deletion and non-coding gene function research, it is often necessary to express two sgRNAs simultaneously. For large-scale application, it is necessary to construct an expression library of sgRNA. A previous study proposed a method for rapidly constructing a Cas9...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N9/22C40B50/06
CPCC12N9/22C12N15/66C40B50/06
Inventor 李大卫汪祖鹏刘义飞钟彩虹黄宏文
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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