Construction and application of CRISPR/Cas9 gene editing vector for microorganisms

A carrier and gene technology, applied in the field of gene editing

Active Publication Date: 2016-01-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is how to construct a single vector for editing Escherichia coli or a single vector for editing the genome of Corynebacterium glutamicum using CRISPR / Cas9 gene editing technology

Method used

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  • Construction and application of CRISPR/Cas9 gene editing vector for microorganisms
  • Construction and application of CRISPR/Cas9 gene editing vector for microorganisms
  • Construction and application of CRISPR/Cas9 gene editing vector for microorganisms

Examples

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Effect test

Embodiment 1

[0064] Example 1. Escherichia coli K-12MG1655 was edited using the Escherichia coli CRISPR / Cas9 gene editing vector

[0065] The Escherichia coli CRISPR / Cas9 gene editing vector consists of the operating region of the arabinose operon, the Cas9 protein gene connected to the operating region, the terminator connected to the Cas9 protein gene to terminate the transcription of the Cas9 protein gene, and the terminator to terminate the transcription of the Cas9 protein gene A linked promoter that initiates the transcription of the DNA encoding the gRNA, a DNA encoding the gRNA linked to a promoter that initiates the transcription of the DNA encoding the gRNA, an origin of replication (including the repA101 gene and the oriR101 gene) linked to the DNA encoding the gRNA, The arabinose operon (including the AraC protein gene and the operating region) linked to the origin of replication, the Red homologous recombination system linked to the arabinose operon, the orf60a gene linked to t...

Embodiment 2

[0085] Example 2: Gene Editing of Corynebacterium glutamicum Using the CRISPR / Cas9 Gene Editing Vector of Corynebacterium glutamicum

[0086] Corynebacterium glutamicum CRISPR / Cas9 gene editing vector consists of a lactose operon, a Cas9 protein gene connected to the lactose operon, a terminator connected to the Cas9 protein gene to terminate the transcription of the Cas9 protein gene, and a terminator to terminate the transcription of the Cas9 protein gene The linked promoter that initiates the transcription of the DNA encoding the gRNA, the DNA encoding the gRNA linked to the promoter that initiates the transcription of the DNA encoding the gRNA, the terminator that terminates the transcription of the DNA encoding the gRNA linked to the DNA encoding the gRNA, and the DNA that terminates the gRNA The per gene linked to the terminator of encoding DNA transcription, the replication initiation site (repA101 protein gene) linked to the per gene, the kanamycin resistance gene linke...

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Abstract

The invention discloses construction and application of a CRISPR/Cas9 gene editing vector for microorganisms. The constructed CRISPR/Cas9 gene vector consists of a replication start site, a selection marker gene, a Cas9 protein gene, gRNA coding DNA, a homologous recombinant element and an operon. The CRISPR/Cas9 gene vector constructed by the invention is capable of editing (including performing such operations as knocking out, replacing, interpolating and the like on gene or DNA sequence) escherichia coli or corynebacterium glutamicum genome; and the gene vector has the advantages of being short in test cycle, time-saving and cost-saving, high in efficiency and the like.

Description

technical field [0001] The invention relates to the construction and application of a CRISPR / Cas9 gene editing vector for microorganisms in the field of gene editing. Background technique [0002] Targeted editing of genes is one of the most important methods in the field of biological research. With the development of science, more and more gene editing technologies have emerged, from the classic EMS random mutagenesis, T-DNA insertion mutagenesis or transposon insertion inactivation to zinc finger nucleases (Zinc-fingernucleases, ZFN) These technologies have greatly promoted the process of gene function research. However, ZFN technology and TALEN technology need to design specific endonucleases for each target gene, and the construction process is cumbersome, which greatly limits their application scope. Compared with other silencing systems, the clustered regularly interspaced short palindromic repeats (CRISPR) technology has its incomparable advantages, and has gradual...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/77C12N15/11
Inventor 毕昌昊张学礼赵东东
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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