Host Cell Recombination Site Layout for Precise Marker Excision
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Solution Overview
Problem
Existing methods struggle to efficiently remove multiple genetic elements from a genome without inadvertently excising required genetic material, particularly when selection markers are positioned closely together, leading to interference during recombination.
Innovation Solution
Utilizing pairs of identical recombination sites with distinct nucleic acid sequences to flank each nucleic acid segment, allowing selective excision of unwanted genetic elements while preserving the genome integrity by using recombinases that recognize these sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard recombination methods are used to remove multiple selection markers, then marker removal efficiency improves, but interference between recombination sites causes unwanted excision of required genetic material
Solution Approach 1:
The patent divides the recombination system into multiple independent segments by using different recombination sites (FRT1, FRT2, FRT3, FRT4) for different selection markers. Each marker is flanked by unique recombination site pairs, allowing independent and specific removal without cross-interference, thus maintaining genome integrity while enabling efficient marker removal.
Solution Approach 2:
The patent applies local quality by making each recombination site pair unique to its specific location and function. FRT1/FRT2 are used for one marker while FRT3/FRT4 are used for another marker, ensuring that recombination events occur only at the intended locations and not elsewhere in the genome, preventing unwanted excisions.
2Productivity
If multiple identical recombination sites are used to flank different genetic elements, then recombination efficiency improves, but cross-reactivity between sites leads to loss of required genetic material
Solution Approach 1:
The patent segments the recombination system by creating distinct recombination site variants (FRT1, FRT2, FRT3, FRT4) that are specific to different genetic elements. This segmentation allows each element to be targeted independently with high precision, eliminating cross-reactivity while maintaining efficient recombination at each site.
Solution Approach 2:
Each recombination site is designed with local quality characteristics unique to its function. The different FRT variants have specific sequence features that make them recognizable only by their corresponding recombinase pairs, ensuring precise excision of the intended genetic element without affecting other regions.
3Quantity of substance
If selection markers are positioned close together in the genome, then genome size is reduced, but recombination interference increases causing excision of required material
Solution Approach 1:
The patent segments the closely positioned selection markers by assigning unique recombination site pairs to each marker. Even though the markers are physically close in the genome, the distinct FRT site sequences create functionally independent recombination zones, preventing interference and ensuring faithful excision of only the intended markers.
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 precise and efficient removal of multiple genetic elements, such as selection markers, from a genome without losing 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
Implementation Method 2
exposing the genomic polynucleotide to a recombinase that recognises the first recombination sites such that the identical recombination sites recombine resulting in the excision of the first insert nucleic acid
Data Source
AI summary
The present invention discloses 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;wherein the first pair of recombination sites have an identical nucleic acid sequence and the second pair of recombination sites have an identical nucleic acid sequence and the first and second pairs of recombination sites share 90-98% nucleic acid sequence identity.Also disclosed is a host cell genome polynucleotide comprising a first recombinantly engineered region and a second recombinantly engineered region, wherein a first single recombination site is adjacent to the first recombinantly engineered region, and a second single recombination site is adjacent to the second recombinantly engineered region, wherein the first and second recombination sites have nucleotide sequences which share 90-98% identity with each other and optionally with the nucleic acid sequence of any further recombination sites present in the host cell genome polynucleotide.