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Temperature-controlled self-cleaving single plasmid homologous recombination system and application thereof in gene editing

A technology of homologous recombination and gene editing, applied in the field of genetic engineering, to avoid accidental rearrangement, save time and cost, and be easy to operate

Pending Publication Date: 2021-10-01
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems that are easy to occur in the process of gene editing using homologous recombination system, the purpose of the present invention is to provide a faster, simpler and more economical homologous recombination system and its construction method

Method used

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  • Temperature-controlled self-cleaving single plasmid homologous recombination system and application thereof in gene editing
  • Temperature-controlled self-cleaving single plasmid homologous recombination system and application thereof in gene editing
  • Temperature-controlled self-cleaving single plasmid homologous recombination system and application thereof in gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: pKID220 plasmid construction

[0045] pKID220 plasmid with temperature control element Cits857-P R P L , to control the sequential expression of I-SceI endonuclease and Red recombinase, the construction process is as follows figure 1 shown. Specific steps are as follows:

[0046] 1. With the pDC plasmid (nucleotide sequence shown in SEQ ID NO.2) as the backbone, the chloramphenicol resistance gene cassette carried by it is flanked by I-SceI endonuclease recognition sites and FLP enzyme recognition targets (FRT). Using the pBV220 plasmid as a template, the 1483bp temperature control element CIts857-P was amplified with pDCprpL-F and pDCprpL-R primers R P L Fragment (sequence shown in SEQ ID NO.3), and the PCR product was cloned into pDC plasmid through Nhe I site to obtain pDC-CItsPRPL plasmid.

[0047] 2. Use primer pair ISceI-F / R and primer pair GBe2-F / R to amplify respectively from pKISa plasmid (nucleotide sequence as shown in SEQ ID NO.5) with codi...

Embodiment 2

[0055] Example 2: Knockout of the pgtE gene of Salmonella enteritidis

[0056] The pKID-UD knockout plasmid is constructed by inserting the flanking DNA sequence carrying the pgtE gene into the targeting fragment region of the pKID220 plasmid, and is used to knock out the pgtE gene from Salmonella enteritidis. Operation process such as image 3 shown.

[0057] The specific operation steps are as follows:

[0058] 1. Use HFF1 and HFR1 primers (shown in Table 1) to amplify the upstream homology arm PgtE of 871bp from the Salmonella enteritidis SM6 strain genome up (sequence shown in SEQ ID NO.6), then use HRF1 and HRR1 primers to amplify the downstream homology arm PgtE of 963bp from SM6 genome down (The sequence is shown in SEQ ID NO.7).

[0059] 2. The pKID220 plasmid was double digested with NcoI and SalI to obtain the chloramphenicol resistance gene fragment FCF (1041bp) with FRT sites on both sides and the backbone fragment CIGBE of 5121bp. Transformation of PgtE by ov...

Embodiment 3

[0063] Embodiment 3: INP0862 gene replaces Salmonella enteritidis pgtE gene

[0064] The pKID-INP0862 knock-in (replacement) plasmid is constructed by inserting the flanking DNA sequence carrying the INP0862 gene into the targeting fragment region of the pKID220 plasmid, and is used to replace the pgtE gene of Salmonella enteritidis with the INP0862 gene. Operation process such as Figure 5 shown. The specific operation steps are as follows:

[0065] 1. Using the pET28a-INP0862 plasmid as a template, use HF0862-F and HF0862-R primers (shown in Table 1) to amplify by PCR to obtain a 1480bp INP0862 gene fragment (sequence shown in SEQ ID NO.8). The One Step Cloning Kit was used to clone into pKID-UD through the Nco I site, and the ligated product was transformed into SM6 strain by electroporation. Specifically, 10 μL of the ligation product was added to the pre-prepared competent cells, and then electroporation was completed by a Bio-Rad micropulse generator (parameters were ...

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Abstract

The invention discloses a temperature-controlled self-cleaving single plasmid homologous recombination system and application thereof in gene editing. The system comprises a single plasmid pKID220 used for gene editing, the pKID220 plasmid contains a temperature control element, a Red recombinase gene, an I-SceI endonuclease gene and a targeting fragment region containing a selection marker, and the two sides of the selection marker are both provided with an I-SceI recognition site and an FRT site. Experiments prove that the recombination efficiency of the homologous recombination system is superior to that of a double plasmid and arabinose induction method of a traditional lambda-Red system, no chemical inducer is needed, and efficient editing of chromosomes can be achieved only by changing the culture temperature. Moreover, multiple electroporation processes are avoided, the operation complexity is greatly reduced, and the time and the cost are saved. The invention provides a quicker, simpler, more convenient and more economical technical means for gene editing.

Description

technical field [0001] The invention relates to a temperature-controlled self-shearing single-plasmid homologous recombination system and its application in gene editing, which belongs to the technical field of genetic engineering. Background technique [0002] It is of great significance to rapidly edit and transform the genome. Homologous recombination is the most widely used gene editing method. The endogenous recombination system and λ-Red recombination system developed based on this principle have been widely used in various modifications of microbial chromosomes, such as Salmonella and Escherichia coli. Most endogenous homologous recombination requires the participation of RecA protein, and usually requires the insertion of hundreds of bases or even longer homologous sequences in the suicide plasmid responsible for targeting. The RecA-mediated reaction is a time-consuming and laborious process, and the recombination efficiency is very low. In addition, the recombinat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/55C12N15/65C12N15/90C12N1/21C12R1/42
CPCC12N15/74C12N9/22C12N15/65C12N15/902C12N2800/30C12N2800/101C12N2800/80Y02A50/30
Inventor 于申业刘思国
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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