CRISPR-Cas9 system for targeting and removing bacterial resistance plasmids and application

Abstract

The invention discloses a gRNA sequence. The sequence in a CRISPR-Cas9 system is capable of editing a DNA sequence by taking a resistance plasmid specific site in bacteria as a target sequence. The gRNA sequence is shown as SEQ ID NO:1 and / or 2. The invention also discloses the CRISPR-Cas9 system of the gRNA sequence and an application of the CRISPR-Cas9 system in preparing a drug for treating resistant bacterial infection. The CRISPR-Cas9 system provided by the invention is capable of targeting two super resistance genes of NDM-1 and MCR-1, simultaneously eliminating the two resistance genes and guaranteeing the use effect of carbapenms and colistin antibiotics.

Description

technical field

[0001] The invention discloses a CRISPR-Cas9 system for targeted removal of bacterial drug-resistant plasmids. Background technique

[0002] Worldwide, 700,000 people die each year from bacterial antibiotic resistance, mostly in Asia and Africa. In China, more than 80,000 people die from drug-resistant bacterial infections every year. According to the estimates of the British Antimicrobial Resistance Assessment Committee, if the current situation cannot be improved, by 2050, 10 million people worldwide will suffer from antimicrobial resistance, which is more than the current number of cancer deaths. The problem of bacterial resistance not only seriously affects human health, but also causes economic losses. A recent report by the World Bank and the Food and Agriculture Organization of the United Nations pointed out that if the problem of antibiotic resistance is not solved by 2050, the global annual GDP will drop by about 1.1%-3.8%, which is equivalent to t...

Examples

Embodiment 1

[0042] Example 1: Construction and screening of a CRISPR-Cas9 system targeting NDM-1 and MCR-1 drug resistance genes using pCas9 as a carrier

[0043] 1. Design of target sequences

[0044] (1) The design of the NDM-1 target sequence, according to the PAM principle in the design requirements of sgRNA, select the 30bp base sequence upstream of the NGG trinucleotide in the blaNDM-1 sequence as the target sequence, that is, the spacer in the CRISPR sequence sequence, which can be transcribed into sgRNA in bacterial cells.

[0045] A high-efficiency and specific target T1 sequence was screened from the twenty initially designed target sequences: SEQ ID NO: 1 (5'-ACCGCATTAGCCGCTGCATTGATGCTGAGC-3').

[0046] (2) Design of MCR-1 drug resistance gene sequence target

[0047] According to the colistin resistance gene MCR-1 sequence, according to the PAM principle, a 30bp base sequence was selected as the target of the gene, SEQ ID NO.2 (5'-atgccctacagaccgaccaagccgagacca-3').

[0048...

Embodiment 2

[0085] Example 2: CRISPR-Cas9 system targeted removal of NDM-1 drug-resistant plasmids

[0086] The CRISPR-Cas9 system plasmid pCas9-N targeting NDM-1 transformed the drug-resistant bacteria model containing the pNDM-1 plasmid. Compared with the control group pCas9, pCas9-N can efficiently remove the natural large plasmid pNDM-1, and can Eliminate high copy NDM-1 cloning plasmids (see image 3 ). The removal efficiencies are all greater than 99.9%. Figure 4 Confocal scanning photos of the removal effect of NDM-1 recombinant EGFP high-copy plasmid, NDM-1 plasmid contains a gene capable of expressing enhanced green fluorescent protein (EGFP), from Figure 4It can be seen that the efficient clearance of NDM-1 by pCas9-N leads to the extreme reduction or even disappearance of EGFP. Figure 5 is the columnar control graph of the fluorescence intensity of the NDM-1 plasmid clearance rate; Figure 5 It can be seen that after pCas9-N plasmid removal, the fluorescence intensity de...

Embodiment 3

[0087] Example 3: CRISPR-Cas9 system pCas9-NM simultaneously targets and eliminates NDM-1 and MCR-1 drug-resistant plasmids

[0088] The NDM-1 and MCR-1 drug-resistant plasmids were cleared by pCas9-NM respectively, and the drug-resistant plasmids of the two drug-resistant bacterial models transformed with the pCas9-NM plasmid were detected. After shearing by pCas9-NM, both drug-resistant plasmids were eliminated and could not be detected by PCR (see Figure 6 ).

[0089] Detect changes in bacterial antibiotic sensitivity after pCas9-NM action. Etest drug-sensitive paper strip quantitatively detects the MIC value of the NDM-1 drug-resistant bacteria model to imipenem (thiamycin antibiotic with a carbapenem ring). / ml is reduced to 0.38ug / ml (see Figure 7 ).

[0090] The plate method was used to qualitatively analyze the changes in the bacterial drug sensitivity results after the MCR-1 plasmid was cleared by pCas9-NM. On the LB plates containing 0.5ug / ml colistin, there w...

technical field

[0001] The invention discloses a CRISPR-Cas9 system for targeted removal of bacterial drug-resistant plasmids. Background technique

[0002] Worldwide, 700,000 people die each year from bacterial antibiotic resistance, mostly in Asia and Africa. In China, more than 80,000 people die from drug-resistant bacterial infections every year. According to the estimates of the British Antimicrobial Resistance Assessment Committee, if the current situation cannot be improved, by 2050, 10 million people worldwide will suffer from antimicrobial resistance, which is more than the current number of cancer deaths. The problem of bacterial resistance not only seriously affects human health, but also causes economic losses. A recent report by the World Bank and the Food and Agriculture Organization of the United Nations pointed out that if the problem of antibiotic resistance is not solved by 2050, the global annual GDP will drop by about 1.1%-3.8%, which is equivalent to t...

Examples

Embodiment 1

[0042] Example 1: Construction and screening of a CRISPR-Cas9 system targeting NDM-1 and MCR-1 drug resistance genes using pCas9 as a carrier

[0043] 1. Design of target sequences

[0044] (1) The design of the NDM-1 target sequence, according to the PAM principle in the design requirements of sgRNA, select the 30bp base sequence upstream of the NGG trinucleotide in the blaNDM-1 sequence as the target sequence, that is, the spacer in the CRISPR sequence sequence, which can be transcribed into sgRNA in bacterial cells.

[0045] A high-efficiency and specific target T1 sequence was screened from the twenty initially designed target sequences: SEQ ID NO: 1 (5'-ACCGCATTAGCCGCTGCATTGATGCTGAGC-3').

[0046] (2) Design of MCR-1 drug resistance gene sequence target

[0047] According to the colistin resistance gene MCR-1 sequence, according to the PAM principle, a 30bp base sequence was selected as the target of the gene, SEQ ID NO.2 (5'-atgccctacagaccgaccaagccgagacca-3').

[0048...

Embodiment 2

[0085] Example 2: CRISPR-Cas9 system targeted removal of NDM-1 drug-resistant plasmids

[0086] The CRISPR-Cas9 system plasmid pCas9-N targeting NDM-1 transformed the drug-resistant bacteria model containing the pNDM-1 plasmid. Compared with the control group pCas9, pCas9-N can efficiently remove the natural large plasmid pNDM-1, and can Eliminate high copy NDM-1 cloning plasmids (see image 3 ). The removal efficiencies are all greater than 99.9%. Figure 4 Confocal scanning photos of the removal effect of NDM-1 recombinant EGFP high-copy plasmid, NDM-1 plasmid contains a gene capable of expressing enhanced green fluorescent protein (EGFP), from Figure 4It can be seen that the efficient clearance of NDM-1 by pCas9-N leads to the extreme reduction or even disappearance of EGFP. Figure 5 is the columnar control graph of the fluorescence intensity of the NDM-1 plasmid clearance rate; Figure 5 It can be seen that after pCas9-N plasmid removal, the fluorescence intensity de...

Embodiment 3

[0087] Example 3: CRISPR-Cas9 system pCas9-NM simultaneously targets and eliminates NDM-1 and MCR-1 drug-resistant plasmids

[0088] The NDM-1 and MCR-1 drug-resistant plasmids were cleared by pCas9-NM respectively, and the drug-resistant plasmids of the two drug-resistant bacterial models transformed with the pCas9-NM plasmid were detected. After shearing by pCas9-NM, both drug-resistant plasmids were eliminated and could not be detected by PCR (see Figure 6 ).

[0089] Detect changes in bacterial antibiotic sensitivity after pCas9-NM action. Etest drug-sensitive paper strip quantitatively detects the MIC value of the NDM-1 drug-resistant bacteria model to imipenem (thiamycin antibiotic with a carbapenem ring). / ml is reduced to 0.38ug / ml (see Figure 7 ).

[0090] The plate method was used to qualitatively analyze the changes in the bacterial drug sensitivity results after the MCR-1 plasmid was cleared by pCas9-NM. On the LB plates containing 0.5ug / ml colistin, there w...

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