Circular Plant Vectors Using eccDNA for Stable Gene Amplification
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Solution Overview
Problem
Current genome engineering technologies for plants face challenges with off-target effects and integration difficulties, limiting the efficient amplification and expression of desired genes for traits such as herbicide resistance.
Innovation Solution
A circular plant vector comprising a first and second tethering nucleic acid, a nucleic acid encoding a polynucleotide of interest, and nucleic acids encoding replicon proteins, which allows for tethering and replication without integration into the genome, leveraging the eccDNA replicon mechanism for stable gene amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current genome engineering technologies are used for plant transformation, then gene transfer can be achieved, but off-target effects and genome integration difficulties occur
Solution Approach 1:
The patent extracts the desired gene amplification function from traditional integration-based transformation systems. By using extrachromosomal circular DNA (eccDNA) that replicates independently without integrating into the plant genome, the system separates gene amplification from genome integration, thereby eliminating off-target integration effects while maintaining reliable gene transfer and amplification
Solution Approach 2:
The patent introduces eccDNA as an intermediary carrier between the transformation vector and the plant genome. This eccDNA acts as a mediator that can be introduced into plant cells, replicate autonomously, and amplify the desired gene without requiring direct integration into chromosomal DNA, thus avoiding off-target integration issues while achieving stable gene expression
2Reliability
If current genome engineering technologies are used for plant transformation, then gene transfer can be achieved, but genome integration difficulties occur
Solution Approach 1:
The patent extracts the gene amplification function from the genome integration process. By designing a system where the transgene is maintained on extrachromosomal circular DNA that replicates independently, the patent eliminates the need for genome integration entirely, making the transformation process simpler and more reliable without the technical challenges of achieving efficient chromosomal integration
Solution Approach 2:
The eccDNA system is designed to be self-replicating within plant cells. The circular DNA contains its own origin of replication and can be amplified autonomously without requiring integration into host chromosomal DNA or dependence on host replication machinery for chromosomal integration, thereby simplifying the transformation process and improving reliability
Data Source
AI summary
The invention relates to a circular vector for plant transformation comprising: a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins and methods for using the same. Also provided are plants produced by the methods of the invention and products produced from the plant.