Nucleic Acid Cassette Removal with Non-Identical Recombination Sites
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods struggle to efficiently remove multiple genetic elements from a genome without inadvertently removing required genetic material, particularly when selection markers are positioned closely together, leading to interference during recombination.
Innovation Solution
Utilizing pairs of non-identical recombination sites to flank each nucleic acid segment to be removed, ensuring that only the intended genetic elements are excised while preserving the rest of the genomic polynucleotide sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If standard recombination methods are used to remove multiple selection markers, then selection markers can be removed from the genome, but interference between recombination sites causes unintended removal of required genetic material
Solution Approach 1:
The patent divides the genome into distinct segments, each flanked by its own unique pair of recombination sites. By using non-identical recombination site pairs (e.g., FRT1, FRT2, FRT3) for different genetic elements, the patent ensures that recombination events are confined to specific segments and do not interfere with other segments. This segmentation approach allows independent removal of multiple selection markers without cross-reactivity between recombination sites.
Solution Approach 2:
The patent applies local quality by making each recombination site pair unique to its specific location and function. Each pair of recombination sites has distinct sequence characteristics that allow selective recognition by specific recombinases. This local differentiation ensures that recombination occurs only at the intended location and not at other sites in the genome, preventing unintended genetic material removal.
2Ease of operation
If multiple identical recombination sites are used to flank different genetic elements, then removal of each element is straightforward, but cross-recombination occurs between sites leading to loss of required genetic material
Solution Approach 1:
The patent introduces asymmetry by using non-identical recombination site pairs for different genetic elements. Instead of using the same recombination site sequence throughout the genome, each genetic element is flanked by a unique pair of recombination sites with distinct sequences. This asymmetric design maintains operational simplicity (each site can still be removed by the same recombinase system) while achieving precision (cross-recombination is prevented due to sequence differences).
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 allows for the precise and efficient removal of multiple genetic elements, such as selection markers, from a genome without disturbing other essential genetic material, facilitating the production of glycosylated proteins and optimizing gene expression.
Implementation Method 1
The Flp recombinase produces recombination between Flp recombinase target (FRT) sites which are genetic elements of about 48 bp
Data Source
AI summary
A process for engineering a host cell comprising the steps of a) integrating a first polynucleotide cassette including a first selection marker flanked by a first pair of recombination sites; b) removing the first selection marker by the action of a recombinase which recognises the first pair of recombination sites; c) integrating a second polynucleotide cassette including a second selection marker flanked by a second pair of recombination sites; and d) removing the second selection marker by the action of a recombinase which recognises the second pair of recombination sites. The first and second pairs of recombination sites have identical nucleic acid sequences within each pair, sharing 90-98% nucleic acid sequence identity. Also disclosed is a host cell genome polynucleotide comprising two recombinantly engineered regions, each adjacent to a single recombination site sharing 90-98% identity with each other and any additional recombination sites present in the host cell genome polynucleotide.