Phage Lambda Integrase Site-Specific DNA Recombination
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Solution Overview
Problem
Current genome engineering methods, such as those using ZFNs, TALENs, and CRISPR/Cas9, lack alternatives for precise and controlled site-specific DNA recombination, limiting the versatility and efficiency of genome modification.
Innovation Solution
A method utilizing phage lambda integrases for site-specific DNA recombination, involving the use of bacterial plasmids with specific nucleotide sequences and intramolecular recombination to integrate a DNA sequence of interest into a target genomic location within a host cell, mediated by phage lambda integrases like Int-h/218 or Int-C3.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If phage lambda integrase is used for site-specific DNA recombination, then integration precision and control are improved, but the versatility of genome engineering is limited compared to other methods
Solution Approach 1:
The patent applies universality by designing a phage lambda integrase system that can perform multiple functions: site-specific integration, excision, and inversion of DNA sequences. The integrase enzyme works with different attP and attB sequences to achieve various genome engineering outcomes, making the system versatile despite using a single enzymatic mechanism.
Solution Approach 2:
The patent utilizes parameter changes by modifying the attP and attB recognition sequences to create different integration specificities. By changing the nucleotide sequences at these sites, the system can target different genomic locations while maintaining precise control through the integrase enzyme.
2Productivity
If intramolecular recombination is used to integrate DNA sequences, then integration efficiency is improved, but bacterial sequence contamination increases
Solution Approach 1:
The patent applies extraction by removing bacterial sequences from the integration construct. The method uses intramolecular recombination between attP sites within the bacterial plasmid to excise the DNA sequence of interest as a circular construct, separating it from bacterial backbone sequences that would otherwise be introduced into the host genome.
Solution Approach 2:
The patent uses segmentation by dividing the bacterial plasmid into separate components through intramolecular recombination. The DNA sequence of interest is excised as a distinct circular molecule, separating it from the bacterial plasmid backbone, which remains as a separate entity and can be eliminated without affecting the integrated sequence.
3Manufacturing precision
If site-specific recombination is used for genome engineering, then control and precision are improved, but the need for alternative technologies remains
Solution Approach 1:
The patent applies the intermediary principle by using phage lambda integrase as a mediator between the DNA sequence of interest and the host genome. The integrase enzyme facilitates controlled recombination between attP and attB sites, providing precise integration while the system can be combined with other genome engineering tools like CRISPR/Cas9 for enhanced versatility.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables stable and precise integration of DNA sequences into specific genomic locations, such as LINE-1 elements, with high efficiency and reduced bacterial sequence contamination, enhancing genome editing capabilities.
Implementation Method 1
subjecting the bacterial plasmid to conditions that allow intramolecular recombination between the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1 and the nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:2 in the presence of a phage lambda integrase
Implementation Method 2
subjecting the host cell to conditions that allow integration of the DNA sequence of interest into the target genomic DNA sequence of the host cell, wherein said integration is mediated by the phage lambda integrase
Data Source
AI summary
The present invention provides a method of stably integrating a DNA sequence of interest into a target genomic DNA sequence of a host cell, wherein the target genomic DNA sequence comprises a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:1, as well as a kit for use in said method. Also provided is a method of generating a circular DNA construct essentially consisting of a DNA sequence of interest and a nucleotide sequence that is at least 80% homologous to the sequence as set forth in SEQ ID NO:3.