Stable Docking Platform for Precise Genome Integration
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Solution Overview
Problem
Current methods for inserting transgenes into the human genome lack precision, often resulting in random integration, disrupting regulatory elements, and limiting the size of DNA that can be inserted.
Innovation Solution
A stable docking platform is developed using a complex strategy combining homologous recombination, site-specific recombination mediated by serine-integrases, and transposon systems to create a serial mechanism for assembling multiple docking modules into a locus of the cell genome, allowing for simultaneous integration of multiple transgenes without the need for nucleases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If programmable nucleases (ZFN, TALEN, CRISPR/Cas9) are used to introduce genetic information, then integration precision is improved, but off-target activity and toxicity increase
Solution Approach 1:
The patent segments the integration process into two independent stages: (1) creating a docking site using programmable nucleases, and (2) integrating transgenes using site-specific recombination. This segmentation allows the precision benefits of nucleases to be isolated to the docking site creation step, while the transgene integration step uses safer recombination enzymes that do not exhibit off-target activity.
Solution Approach 2:
The patent introduces a docking site as an intermediary element that mediates between the programmable nuclease system and the transgene integration. The docking site serves as a stable platform that can be created once with high precision, and then used repeatedly for safe transgene integration without requiring repeated nuclease activity, thereby eliminating ongoing off-target risks.
2Object-affected harmful factors
If site-specific recombination with SSRs is used, then off-target activity is reduced, but requirement for pre-existing attachment sites limits versatility
Solution Approach 1:
The patent performs preliminary action by creating the docking site attachment site in advance using programmable nucleases. This pre-established docking site then serves as the foundation for subsequent site-specific recombination events, allowing the system to combine the versatility of nuclease-mediated targeting with the safety of recombination-based integration.
3Productivity
If multiple transgenes are integrated simultaneously, then productivity is improved, but complexity of integration system increases
Solution Approach 1:
The patent creates a universal docking site that can accommodate multiple different transgenes through sequential site-specific recombination events. The docking site serves as a multi-functional platform that simplifies the integration process, as the same docking site structure can receive various transgenes without requiring complex differentiating features for each integration event.
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 and precise integration of genetic material, reducing the risk of off-target events and allowing for the sequential addition of new docking sites, facilitating the study of metabolic routes and gene interactions.
Implementation Method 1
integrating a nucleotide sequence A into the target gene from an isolated cell genome by homologous recombination
Implementation Method 2
site-specific recombination mediated by serine-integrases
Implementation Method 3
catalyzed by a phage-encoded integrase protein (Int)
Implementation Method 4
Transposable elements are DNA sequences that move from one location on the genome to another. Transposon DNA has Inverted Terminal Repeats (ITRs) that are recognized by specific transposases and moved by cut and paste mechanism
Data Source
AI summary
The present invention relates to a method for integrating one or more recombinant sites in a sole target gene of a cell genome comprising carrying out sequentially multiple site-specific recombination by using alternatively two different serine-integrases, wherein the selection marker is removed after each site-specific recombination by different transposases: An in vitro method for inserting at least one recombinant site in a target gene/locus from an isolated cell genome, said method comprising integrating a nucleotide sequence A into the target gene from an isolated cell genome by homologous recombination, wherein said nucleotide sequence A comprises a recombination cassette, flanked at 5′ and 3′ by target gene homology arms, and removing the selection marker by using the second transposase, thus obtaining a cell genome A comprising (i), (ii) and (iii) of nucleotide sequence A.