Serine Recombinases for Stable Eukaryotic Gene Integration
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Solution Overview
Problem
Current site-specific recombination systems in eukaryotic cells, such as the Cre-loxP and FLP-FRT systems, are reversible and limited in the number of recombination events, leading to undesired chromosomal rearrangements and instability in gene integration and expression.
Innovation Solution
Employing novel prokaryotic recombinases like A118, SF370.1, SPβc2, φRv1, and Bxb1, which mediate unidirectional recombination between specific attachment sites (attB and attP) in eukaryotic cells, ensuring stable and irreversible integration or excision of polynucleotides, preventing reverse reactions and chromosomal rearrangements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If reversible recombination systems like Cre-loxP and FLP-FRT are used, then flexibility in recombination events is improved, but chromosomal stability deteriorates due to undesired rearrangements and reversibility
Solution Approach 1:
The patent inverts the traditional reversible recombination approach by using serine recombinases that catalyze unidirectional recombination reactions. Instead of allowing reverse reactions as in Cre-loxP systems, the serine recombinase system proceeds in one direction only, converting the reversible process into an irreversible one, thereby ensuring chromosomal stability while maintaining recombination flexibility through enzyme selection
Solution Approach 2:
The patent changes the fundamental parameter of recombination reversibility by switching from tyrosine recombinases (Cre, FLP) that enable reversible reactions to serine recombinases that enforce unidirectional reactions. This parameter change from reversible to irreversible chemistry resolves the contradiction by eliminating chromosomal rearrangements while preserving the ability to perform targeted recombination events
2Adaptability or versatility
If multiple recombination events are performed with the same recombinase system, then genetic manipulation versatility is improved, but recombination efficiency deteriorates due to limited reaction capacity
Solution Approach 1:
The patent segments the recombination system into multiple independent serine recombinase systems, each with its own specific att sites. This allows different serine recombinases to be used in sequence or parallel without cross-reactivity, enabling multiple recombination events to occur efficiently without the diminishing returns seen in single-system approaches
Solution Approach 2:
The patent introduces specific att sites as intermediaries that mediate between the recombinase enzyme and the DNA substrate. These specialized attachment sites enable each serine recombinase to recognize and act on its specific targets, facilitating multiple sequential recombination events while maintaining high efficiency for each individual reaction
3Manufacturing precision
If site-specific recombination is performed in eukaryotic cells, then gene integration precision is improved, but system availability deteriorates due to limited recombinase options
Solution Approach 1:
The patent demonstrates that serine recombinases, originally derived from prokaryotic systems, can function universally in eukaryotic cells. By showing that these enzymes maintain their site-specific recombination capability across different cellular environments, the patent expands the available toolset for eukaryotic genetic manipulation while preserving integration precision
Solution Approach 2:
The patent uses att sites as specialized intermediaries that bridge the compatibility gap between prokaryotic serine recombinases and eukaryotic cellular environments. These attachment sites enable the foreign enzymes to function precisely in eukaryotic cells, effectively expanding system availability without compromising integration precision
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
This approach enables stable, site-specific recombination in eukaryotic cells, allowing for precise integration, excision, inversion, and translocation of polynucleotides, enhancing the stability and control of transgene expression and chromosome rearrangements without inducing unwanted chromosomal changes.
Implementation Method 1
a recombinase enzyme, which catalyzes the recombination event, and two recombination sites
Data Source
AI summary
The present invention provides a method for obtaining site-specific recombination in a eukaryotic cell, the method comprising providing a eukaryotic cell that comprises a first recombination attachment site and a second recombination attachment site; contacting the first and second recombination attachment sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination attachment sites, wherein the recombinase polypeptide can mediate recombination between the first and second recombination attachment sites, the first recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB), the second recombination site is attB or attP, and the recombinase is selected from the group consisting of a Listeria monocytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase and a Mycobacterium smegmatis phage recombinase, provided that when the first recombination attachment site is attB, the second recombination attachment site is attP and when the first recombination attachment site is attP, the second recombination attachment site is attB. The invention also describes compositions, vectors, and methods of use thereof, for the generation of transgenic cells, tissues, plants, and animals. The compositions, vectors and methods of the present invention are also useful in gene therapy applications.