Method for knocking out drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof

A drug resistance gene, mcr-1 technology, applied in the field of microbial genetic engineering and biology, to achieve the effect of broad application prospects

Active Publication Date: 2017-03-29
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after knocking out the drug-resistant gene mcr-1 in vitro, the method of delivering the deleted gene into cells by cell-penetrating peptides has not been reported yet.

Method used

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  • Method for knocking out drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof
  • Method for knocking out drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof
  • Method for knocking out drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of the CRISPR-Cas9 system for the mcr-1 gene

[0048] 1. Compare the mcr-1 gene sequence to find a relatively conserved region. The mcr-1 gene-specific targeting sequence in this region is shown in SEQ ID NO. 1. The targeting sequence is designed with sgRNA and a sgRNA, its corresponding DNA sequence information is shown in SEQ ID NO.5.

[0049] 2. Construction of pCas9-mcr:

[0050] (1) Design and synthesize the sgRNA DNA sequence oligo DNA that recognizes the mcr-1 gene according to SEQ ID NO.5;

[0051] (2) Perform gradient cooling and annealing on the synthesized oligo DNA sequence, the specific steps are:

[0052] Mix the synthesized oligo DNA (100 μM, 1 μL) with 10xPCR Buffer (1 μL), then add 10 μL of water to make up the system; then denature at 95°C for 5 minutes, and then cool down to 25°C at a rate of 2°C per minute to complete the reaction; anneal After that, a synthetic oligo DNA hybrid duplex is formed.

[0053] (3) Use BsaI to di...

Embodiment 2

[0063] Example 2 Penetrating peptide CPP5a in Staphylococcus aureus JS17

[0064] The test steps are as follows:

[0065] 1. Synthesize the cell penetrating peptide CPP5a according to SEQ ID NO.2, and use Alexa Fluor R 488 ProteinLabeling Kit for labeling (the operation steps were carried out in strict accordance with the instructions), and the protein after labeling was named CPP5a-488; the labeling compound can be combined with the N-terminal NH of the protein 2 - The residues are linked so that the labeled protein emits green fluorescence under laser excitation.

[0066] 2. Add Staphylococcus aureus JS17 to the TSB medium, and then add CPP5a-488 at a final concentration of 0.1 mg / ml, co-cultivate at room temperature for 1 hour, and wash off the recombinant protein with PBS.

[0067] 3. Take the MAC-T cells infected with Staphylococcus aureus JS17 co-cultured in step (2) for 1 hour as the experimental group (GFP-CPP5a), and set up MAC-T cells infected with Staphylococcus...

Embodiment 3

[0068] Example 3 Gel retardation experiment of penetrating peptide CPP5a and sgRNA-Cas9 expression vector pCas9-mcrDNA The binding of cell penetrating peptide CPP5a to DNA was analyzed by electrophoretic mobility. Migration is affected, ie there is a lag compared to the distance that DNA alone migrates.

[0069] Experimental group (12 μL): DNA of vector pCas9-mcr 250 ng, vector pCas9-mcr obtained in step (4) of Example 1 45 μg, penetrating peptide obtained in step (5) of Example 1 45 μg, ddH 2 0 to make up 12 μL;

[0070] Control group (12 μL): DNA of vector pCas9-mcr 250 ng, vector pCas9-mcr obtained in step (4) of Example 1 45 μg, ddH 2 0 to make up 12 μL;

[0071] The control group and the experimental group were incubated at room temperature for 30 minutes, respectively. After incubation, run electrophoresis with 0.8% agarose gel at a voltage of 120V. After electrophoresis, place the electrophoresis gel in a gel imaging system, observe and record under ultraviolet light...

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Abstract

The invention provides a method for knocking out a drug resistance gene mcr-1 through CRISPR-Cas9 in vitro and specialized cell penetrating peptide thereof. The method comprises the steps that a CRISPR-Cas9 system is constructed according to a target point sequence of an mcr-1 drug resistance gene sgRNA identification zone, a base sequence of the target point sequence is shown as SEQ NO.1, in the constructed CRISPR-Cas9 system, a T7 promoter is inserted into the portion in front of a start point of sgRNA transcription, a prokaryotic expression vector of Cas9 protein is constructed, regulation is conducted through the T7 promoter, the specialized cell penetrating peptide CPP2a is utilized for knocking out the mcr-1 drug resistance gene, a carrier pCas9-mcr is brought into a microbial cell, the microbial species can be gram negative bacteria, and the phenomenon that microbial mcr-a drug resistance gene is knocked out in vitro is achieved.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering and biotechnology, in particular to a method for knocking out microbial drug resistance gene mcr-1 in vitro using a CRISPR-Cas9 system and its special cell-penetrating peptide CPP5a. Background technique [0002] Professor Liu Jianhua of South China Agricultural University and Professor Shen Jianzhong of China Agricultural University found a new gene mcr-1 of polymyxin resistance in animals and hospitalized patients (Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, SpencerJ, Doi Y , Tian G, Dong B, Huang X, Yu LF, Gu D, Ren H, Chen X, Lv L, He D, Zhou H, Liang Z, Liu JH, Shen J. 2016. Emergence of plasma-mediated colistin resistance mechanism MCR -1 in animals and human beings in China: amicrobiological and molecular biological study. Lancet Infect Dis 16:161-168.) This gene is carried by a plasmid and can be transferred horizontally between different strains. Before the publication of this d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K7/06
Inventor 孙利厂王冉张莉莉庞茂达何涛
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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