Fluorous Complex Delivery of Genome-Editing Agents to Plant Cells
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Solution Overview
Problem
Existing plant transformation methods require tissue culture through a dedifferentiated callus phase, are limited to specific plant species or genotypes, and involve regulatory hurdles, leading to unintended phenotypic changes and somaclonal variation.
Innovation Solution
A method using a complex of biological materials and a fluorous agent, such as C9H5F15O with a fluoro-surfactant, to deliver genome-editing agents directly to plant cells without requiring tissue culture, allowing for non-random genomic modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If tissue culture through callus phase is used for plant transformation, then transformation can be achieved, but unintended phenotypic changes and somaclonal variation occur
Solution Approach 1:
The patent extracts and eliminates the callus phase from the transformation process. By using direct delivery methods (electroporation, microinjection, or particle bombardment) to introduce genome-editing agents directly into plant cells, the technology bypasses tissue culture through callus, thereby preventing the somaclonal variation and unintended phenotypic changes that occur during callus formation and regeneration.
Solution Approach 2:
The patent introduces genome-editing agents (such as CRISPR-Cas systems, zinc-finger nucleases, or TALENs) as intermediaries to achieve precise genetic modification without requiring tissue culture. These agents enable direct in vivo editing of the plant genome, eliminating the need for dedifferentiated callus phase and subsequent regeneration, thus avoiding somaclonal variation while maintaining transformation effectiveness.
2Reliability
If Agrobacterium tumefaciens-mediated transformation is used, then DNA transfer can be achieved, but integration of T-DNA sequences and regulatory hurdles occur
Solution Approach 1:
The patent extracts only the essential function of Agrobacterium (DNA delivery capability) while eliminating the harmful T-DNA integration mechanism. By using engineered genome-editing agents delivered through physical or chemical methods, the technology achieves precise genetic modification without integrating foreign bacterial DNA sequences into the plant genome, thereby avoiding the regulatory hurdles and genetic contamination associated with Agrobacterium-mediated transformation.
Solution Approach 2:
The patent replaces the biological mechanism of Agrobacterium (which relies on natural transformation and T-DNA integration) with physical or chemical delivery methods (electroporation, microinjection, or particle bombardment). This substitution eliminates the need for bacterial vectors and T-DNA integration, allowing direct introduction of genome-editing components that do not integrate into the plant genome, thus avoiding the harmful effects of foreign DNA integration.
3Reliability
If antibiotic or herbicide resistance transgenes are used for selection, then transformed plants can be identified, but stringent regulatory requirements are imposed
Solution Approach 1:
The patent extracts the selection function from antibiotic or herbicide resistance markers. By using genome-editing agents that create precise, targeted modifications, the technology enables selection of edited plants through molecular detection methods (such as PCR or sequencing) rather than phenotypic selection with resistance markers. This eliminates the need for transgenic selection markers and the associated regulatory burden of releasing genetically modified organisms with foreign genes.
Solution Approach 2:
The patent uses molecular copying and detection methods (such as PCR amplification and sequence analysis) to identify transformed plants without requiring antibiotic or herbicide resistance transgenes. These detection methods copy and analyze the specific genome-editing signatures (such as indels or precise insertions) to confirm transformation, providing a transgene-free selection approach that reduces regulatory complexity while maintaining reliable identification of edited plants.
4Ease of manufacture
If methods limited to specific plant species are used, then transformation protocol can be simplified, but versatility across plant species is reduced
Solution Approach 1:
The patent employs genome-editing agents (such as CRISPR-Cas systems, zinc-finger nucleases, or TALENs) that can be designed and delivered to a wide range of plant species through universal delivery mechanisms (electroporation, microinjection, or particle bombardment). The modular nature of these editing systems allows adaptation to different plant genomes by simply changing the guide sequences or nuclease specificity, providing a versatile platform that maintains protocol simplicity while achieving broad species compatibility across monocots and dicots.
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 efficient delivery of genome-editing agents to various plant species without tissue culture, reducing regulatory burdens and minimizing unintended genetic changes.
Implementation Method 1
contacting the plant cell with a complex comprising the biological material and a fluorous agent
Data Source
AI summary
Provided herein is a method of delivering a biological material, including a genome-editing agent, to the interior of a plant cell wherein the method includes contacting the plant cell with a complex comprising the biological material and a fluorous agent. Also provided herein are compositions and reagents for practicing the method.