Panto Recombinase for Site-Specific DNA Recombination
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Solution Overview
Problem
Current site-specific DNA recombination systems are not equally effective across various cell types and can exhibit off-target activity, limiting their applicability in complex genetic studies and applications requiring simultaneous use of multiple recombinases.
Innovation Solution
Development of a novel recombinase system, Panto, with a protein exhibiting high amino acid sequence identity to WP_008927019.1, which recognizes specific 'pox' sites for site-specific DNA recombination, allowing for efficient and specific genetic manipulations in diverse cell types with low toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing site-specific DNA recombination systems (Cre/loxP, Flp/FRT) are used, then genetic manipulation can be performed in prokaryotic and eukaryotic organisms, but the systems show different activities in cells of different origin and exhibit off-target activity, limiting their applicability across diverse cell types
Solution Approach 1:
The invention divides the existing recombinase systems into separate functional components and creates a novel Panto/pox system with distinct recognition specificity. The Panto recombinase is engineered to recognize unique pox sites that are different from loxP, FRT, rox, and other known recognition sites, thereby segmenting the genetic manipulation toolkit into non-cross-reactive modules that can be independently applied across different cell types without off-target effects
Solution Approach 2:
The invention changes the fundamental parameters of the recombinase system by introducing a novel amino acid sequence (WP_008927019.1) with different recognition specificity. The Panto recombinase recognizes pox sites with a specific nucleotide sequence that differs from all previously used recognition sites, fundamentally altering the system's specificity parameter and enabling reliable use across diverse cell types including mammalian cells where existing systems fail
2Adaptability or versatility
If multiple recombinase systems are used simultaneously for complex genetic studies, then more genetic manipulations can be performed, but the complexity of choosing optimal recombinases for each organism and avoiding cross-reactivity increases
Solution Approach 1:
The Panto/pox system is designed as a universal recombinase module that can function across all cell types (prokaryotic and eukaryotic) without requiring optimization for each organism. The system's universal compatibility and distinct recognition specificity allow it to be used simultaneously with other recombinase systems (Cre/loxP, Flp/FRT, Dre/rox) in the same experimental setup without cross-reactivity, simplifying the management of multiple recombinases by providing a standardized, non-conflicting option
Solution Approach 2:
The Panto recombinase acts as an intermediary tool that mediates genetic manipulations at unique pox sites that do not overlap with other recognition sites. This intermediary function allows researchers to perform multiple independent genetic modifications using different recombinase systems in the same organism without interference, as each system operates on its own distinct recognition sequence
3Reliability
If existing recombinase systems are used in cell types where they show low activity or high toxicity, then genetic manipulation may fail or cause harmful effects, but finding alternative systems for each cell type increases the complexity of experimental design
Solution Approach 1:
The Panto recombinase is engineered as a universal solution that maintains high activity and low toxicity across all cell types including mammalian cells, bacterial cells, and other organisms where existing systems fail. The system's broad compatibility eliminates the need to select different recombinases for different cell types, as Panto/pox provides consistent reliable performance universally
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
Panto enables robust and specific site-specific DNA recombination in various cell types, including bacterial and mammalian cells, with high activity and low toxicity, making it suitable for applications where other systems fail, such as in producing transgenic animal models and biomedical research.
Implementation Method 1
a protein with recombinase activity, wherein the protein comprises an amino acid sequence exhibiting at least 70 %, preferably at least 80 %, preferably at least 90 %, particularly preferred at least 95 %, even more preferred at least 99 % amino acid sequence identity to SEQ ID No. 35 to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other
Data Source
Figure 1
AI summary
The invention relates to the use of a protein with recombinase activity to catalyze a site-specific DNA recombination and a method for producing a site-specific DNA recombination. The invention is applicable alone or in combination with other recombinase systems for genetic manipulation, for example in medical research. The objective of the invention is solved by the use of a protein with recombinase activity to catalyze a site-specific DNA recombination at, preferably at two, recognition sites that are identical or reverse complementary to each other. The invention also includes a method for producing a site-specific DNA recombination comprising the steps of a) providing a cell comprising at least two recognition sites that are identical or reverse complementary to each other; and b) contacting a protein with recombinase activity with the recognition sites, thereby producing the site-specific DNA-recombination.