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
CN106544351AActive Publication Date: 2017-03-29JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
Publication Date
2017-03-29

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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.
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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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