Method of packaging CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using temperate phage vector

A bacteriophage, mild technology, applied in biochemical equipment and methods, other methods of inserting foreign genetic material, stably introducing foreign DNA into chromosomes, etc. The effect of slowing transmission, blocking horizontal transfer, and reducing drug resistance

Active Publication Date: 2017-11-21
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is more stable in the application of wild phage, but the operation steps are relatively cumbersome and time-consuming, and cannot be supplied in time for clinical or other applications.
Recombination of large fragments often requires the assistance of antibiotic tags, but the process of deleting the resistance tag after recombination requires secondary recombination, which is ...

Method used

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  • Method of packaging CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using temperate phage vector
  • Method of packaging CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using temperate phage vector
  • Method of packaging CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using temperate phage vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Construction and screening of a CRISPR-Cas9 system targeting NDM-1 and MCR-1 drug resistance genes using pCas9 as a carrier

[0050] 1. Design of target sequences

[0051] (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.

[0052] A high-efficiency and specific target T1 sequence was screened from the twenty initially designed target sequences: SEQ ID NO: 1 (5'-ACCGCATTAGCCGCTGCATTGATGCTGAGC-3').

[0053] (2) Design of MCR-1 drug resistance gene sequence target

[0054] 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').

[0055...

Embodiment 2

[0093] Example 2: CRISPR-Cas9 system targeted removal of NDM-1 drug-resistant plasmids

[0094] 1. The CRISPR-Cas9 system plasmid pCas9-N targeting NDM-1 was transformed into a drug-resistant bacterial model containing the pNDM-1 plasmid. Efficient removal of 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 4 It 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 decreased by more than 600 times.

Embodiment 3

[0095] Example 3: CRISPR-Cas9 system pCas9-NM simultaneously targets and eliminates NDM-1 and MCR-1 drug-resistant plasmids

[0096] 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 ).

[0097] 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 ).

[0098] 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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Abstract

The invention discloses a method of packaging a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat-associated 9) system by using a temperate phage vector. The method comprises the steps of: (1) constructing suicide genes, target spot sequences bound with specific gRNA (guide ribonucleic acid), and downstream PAM (protospacer adjacent motif) sequences into pSTK (protein serine threonine kinase) plasmids, (2) transforming the pSTK plasmids into escherichia coli host bacteria, (3) transforming CRISPR-Cas9 sequence recombination template double-chain DNA (deoxyribonucleic acid) linear fragments carrying phage sequence homologous arms at the two ends into the host bacteria, (4) inducing expression of homologous recombination related enzymes and the suicide genes SacB, (5) screening the host bacteria subjected to homologous recombination, and (6) inducing temperate phages to crack the host bacteria, and harvesting the recombined temperate phages packaging the CRISPR-Cas9 system, wherein chromosomes of the escherichia coli host bacteria are integrated with the temperate phages; and plasmids capable of expressing the homologous recombination related enzymes are transformed into the escherichia coli host bacteria. According to the packaging method, a secondary recombination step of deleting a resistance marker is removed, and the technical support is provided for the phage vector presenting CRISPR-Cas9 system to resist drug-resistance bacteria efficiently and quickly.

Description

technical field [0001] The invention discloses a phage packaging method. 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. Antibiotic abuse 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 the impact of the 2008 financial crisis. What's more serious is that the ...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/902
Inventor 宋宏彬邱少富刘鸿博李浩梁媛杨超杰赵荣涛贾雷立李鹏王立贵
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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