Polyploid Genome Editing With Markerless Recombination
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Solution Overview
Problem
Current genetic engineering techniques for polyploid organisms, such as cyanobacteria, are hindered by the lack of shuttle vectors and counter-selection systems, making it difficult to achieve efficient and markerless genetic modifications.
Innovation Solution
A system comprising nucleic acid constructs with site-specific recombination systems, such as Cre-LoxP, is used to integrate and excise reporters and modifications in essential genes, allowing for markerless genetic modifications and full segregation of nucleic acid sequences in polyploid organisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional counter-selection systems (e.g., sacB) are used for genetic engineering, then selection markers can be removed, but the system cannot be applied to polyploid organisms like cyanobacteria due to lack of compatibility
Solution Approach 1:
The patent creates a universal genetic engineering system that works across multiple polyploid organisms (cyanobacteria, plants, algae) by using homologous recombination-based counter-selection that exploits polyploidy itself as the selection mechanism, rather than relying on organism-specific markers. The system can be applied to any polyploid organism regardless of its specific biological characteristics.
Solution Approach 2:
The system uses the polyploid nature of the organism itself as the selection mechanism. By introducing a construct with a single copy of a marker gene into a polyploid organism, the system leverages the organism's own genetic architecture (having multiple copies of essential genes) to achieve marker loss through cell division, making the organism's polyploidy the tool for selection rather than an obstacle.
2Ease of manufacture
If shuttle vectors are used for genetic modification, then DNA can be delivered into host organisms, but the vectors are not available or compatible for many polyploid organisms
Solution Approach 1:
The patent extracts and eliminates the need for shuttle vectors by using a direct homologous recombination approach. Instead of delivering DNA via plasmid vectors that must be removed later, the system uses linear DNA constructs with homology arms that directly integrate into the genome through homologous recombination, simplifying the delivery and selection process.
Solution Approach 2:
The patent introduces an intermediary mechanism - homologous recombination - that facilitates DNA integration without requiring vector systems. The homology arms act as intermediaries that guide the DNA construct to the target locus and mediate its integration through natural recombination processes, bypassing the need for vector-based delivery systems.
3Manufacturing precision
If multiple steps and genetic parts are used for DNA modification, then modifications can be achieved, but the process becomes complex and time-consuming
Solution Approach 1:
The patent merges multiple functions into a single construct: the DNA modification function, the counter-selection function, and the marker loss function are all integrated into one homology-based construct. This eliminates the need for separate vector delivery, separate marker removal steps, and separate selection processes, reducing the overall complexity while maintaining precision.
Solution Approach 2:
The system performs preliminary action by incorporating the counter-selection marker and homology arms into the DNA construct before transformation. This pre-prepared construct directly enables both the DNA modification and the subsequent marker loss in a single integrated process, eliminating the need for multiple sequential steps.
4Adaptability or versatility
If plasmids with large size (>10 kb) are used, then broad host range is achieved, but transformation efficiency decreases and handling becomes difficult
Solution Approach 1:
The patent segments the genetic information into smaller, more manageable pieces by using linear DNA constructs with homology arms rather than large circular plasmids. The essential functions are distributed across multiple smaller elements (homology arms, marker gene, selection cassette) that can be more easily handled and transformed, improving transformation efficiency while maintaining host range through the versatility of the homologous recombination mechanism.
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
The system enables efficient, markerless genetic modifications in polyploid organisms by exploiting homologous recombination and site-specific recombination, ensuring all copies of the target nucleic acid site are modified without residual selection markers.
Implementation Method 1
The first construct further comprises regions of homology to a first locus in an essential nucleic acid sequence in the organism flanking the nucleic acid modification system and the first reporter for integration of the first nucleic acid construct into the locus
Implementation Method 2
A system comprising nucleic acid constructs with site-specific recombination systems, such as Cre-LoxP, is used to integrate and excise reporters and modifications in essential genes
Data Source
AI summary
Compositions and methods for genetically modifying at least one nucleic acid sequence of interest in a polyploid organism.