pCasSA plasmid and application thereof

A plasmid and golden yellow technology, applied in the field of pCasSA plasmid, can solve the problem of genome editing without Staphylococcus aureus, achieve high-efficiency transcription inhibition and broad application prospects

Active Publication Date: 2017-06-13
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] CRISPR / Cas9 genome editing technology has achieved efficient genome editing in mammalian cells and some model organisms, such as Escherichia coli, yeast, etc., but has not yet achieved genome editing in Staphylococcus aureus

Method used

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  • pCasSA plasmid and application thereof
  • pCasSA plasmid and application thereof
  • pCasSA plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: pCasSA plasmid construction:

[0033] The composition of the pCasSA plasmid is attached figure 1Shown, its sequence is SEQ ID NO:1. The specific construction method of the pCasSA plasmid is as follows:

[0034] (1) The genome of Staphylococcus aureus Newman strain was extracted using the Ezup Column Bacterial Genomic DNA Extraction Kit produced by Sangon Bioengineering (Shanghai) Co., Ltd., and the specific steps were carried out according to the operation manual of the kit. The promoter of the rpsL gene was amplified by PCR using the genome of the Newman strain as a template; the Cas9 protein gene was amplified by PCR from the pCas9 plasmid; the temperature-sensitive repF replicon and the chloramphenicol-resistant gene were amplified by PCR respectively from the pKOR1 plasmid. Sex gene fragment; DNA fragment containing ColE1 replicon and kanamycin resistance gene was obtained by PCR amplification from pCRISPR plasmid.

[0035] The primer sequences used...

Embodiment 2

[0068] Example 2: pCasSA achieves efficient gene knockout in various strains of Staphylococcus aureus:

[0069] The pCasSA plasmid can be used to efficiently knock out different genes in various strains of Staphylococcus aureus. In the experiment, the agrA gene was selected as an example, and gene knockout experiments were carried out in Staphylococcus aureus RN4220, Newman and USA300 strains. attached figure 2 A is a schematic diagram of gene knockout in Staphylococcus aureus using pCasSA system, and PCR verification, hemolysis experiment and DNA sequencing results of agrA gene knockout in Newman strain.

[0070](1) First select a DNA fragment of the first 20 bases of a certain NGG (N is any base) sequence on the target gene (these 20 bases are called spacers, NGG is not included in it, and the GC ratio of the spacer Preferably at 40%-60%). The feature of this step is that in order to enable the spacer fragment to be inserted into the pCasSA plasmid, GAAA needs to be adde...

Embodiment 3

[0108] Example 3: pCasSA achieves efficient single-base mutations in various strains of Staphylococcus aureus:

[0109] The pCasSA plasmid can be used to perform highly efficient single-base mutations in different genes in various strains of Staphylococcus aureus. In the experiment, the agrA gene was selected as an example, and the gene single base mutation experiment was carried out in Staphylococcus aureus RN4220 and Newman strains. attached figure 2 B is a schematic diagram of using the pCasSA system to carry out single-base mutation of the gene in Staphylococcus aureus, and the hemolysis experiment and DNA sequencing results of the single-base mutation of the agrA gene in the Newman strain.

[0110] (1) Select the spacer of the target gene, and design primers to construct the plasmid. In the experiment, the guanine G at position 94 in the agrA gene was selected and mutated into thymine T. After the mutation, the TGG on the original agrA gene becomes TGT, and the Cas9 p...

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Abstract

The invention provides pCasSA plasmid and an application thereof. The sequence of the pCasSA plasmid is as shown in SEQ ID NO: 1. The pCasSA plasmid is capable of (1) effectively and rapidly editing genomes of various staphylococcus aureus strains, including gene knockout, single base mutation and gene insertion, and (2) having an efficient transcription inhibition function on target genes. The technique has wide application prospects in such aspects as staphylococcus aureus infection treatment, medicine target discovery, medicine development and staphylococcus aureus physiological study.

Description

technical field [0001] The invention relates to a pCasSA plasmid and its application. Background technique [0002] Staphylococcus aureus is an extremely important positive human pathogenic bacteria. It can infect almost any part of the human body, ranging from mild superficial skin infections to fatal severe infections such as toxic shock syndrome, necrotizing pneumonia, and endocarditis. The strong infection and pathogenicity of Staphylococcus aureus are mainly attributed to the secreted cytotoxins and various multifunctional immune manipulation proteins on the cell surface, including aggregation factors, fibronectin-binding proteins, and collagen-binding proteins. These surface proteins, by directly interacting with human cells, can evade host immune response, biofilm formation, bacterial colonization and other aspects to facilitate infection. In recent years, the large-scale prevalence of drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N1/21C12R1/445C12R1/19
CPCC07K14/31C12N15/65C12N15/74C12N2810/10
Inventor 季泉江陈未中
Owner SHANGHAI TECH UNIV
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