Pollen-Specific ZFN Transgene Excision
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Solution Overview
Problem
Current strategies for preventing gene flow from genetically modified (GM) crops to non-GM crops and wild species are limited, particularly in ensuring the excision of transgenes from pollen, which poses risks of ecological contamination and trade restrictions.
Innovation Solution
The use of zinc finger nucleases (ZFNs) linked to pollen-specific promoters to specifically excise transgenes from pollen, eliminating residual recognition sequences and reducing gene flow, thereby enhancing biocontainment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If transgenes are introduced into plants to improve agricultural value, then desirable traits are achieved, but transgene escape to wild species and non-transformed species occurs through gene flow
Solution Approach 1:
The patent applies preliminary action by introducing a pollen-specific excision system before transgene escape can occur. The excision cassette is designed to be expressed specifically in pollen, proactively removing transgenes at the source of gene flow (pollen) before they can be transferred to wild species or non-transformed crops through cross-pollination.
Solution Approach 2:
The patent applies local quality by making the excision system tissue-specific to pollen only. The pollen-specific promoter ensures that the excision cassette is activated exclusively in pollen grains, leaving transgenes intact in other plant tissues. This localized approach allows the plant to maintain desirable traits in vegetative parts while eliminating transgenes from reproductive cells.
2Reliability
If selectable marker genes are used to identify transformed plants, then stable transformation is achieved, but transgene contamination of non-GM varieties occurs through cross-pollination
Solution Approach 1:
The patent applies preliminary action by pre-equipping transformed plants with a pollen-specific excision system that will automatically remove selectable marker genes and other transgenes from pollen before pollination occurs. This proactive measure ensures that even though the plant is stably transformed (maintaining desirable traits), the pollen it produces is free of transgenes, preventing contamination of non-GM varieties.
Solution Approach 2:
The patent applies local quality by restricting the excision activity to pollen tissue only. The pollen-specific promoter drives expression of the excision cassette exclusively in male gametophytes, while selectable marker genes remain functional in other plant tissues for identification and selection purposes during breeding.
3Productivity
If transgenes are introduced to confer resistance traits, then crop improvement is achieved, but ecological risks arise if transgenes persist and establish in weedy populations
Solution Approach 1:
The patent applies preliminary action by implementing a pollen-specific excision mechanism that removes transgenes before they can be transferred to weedy populations through natural cross-pollination. The excision system is activated in pollen grains, proactively preventing the establishment of transgenic weed populations while the crop plants themselves maintain improved traits during their growth cycle.
Solution Approach 2:
The patent applies local quality by making the transgene persistence localized to vegetative tissues only. The pollen-specific promoter ensures that excision occurs exclusively in reproductive cells (pollen), allowing the plant to display improved traits in leaves, stems, and roots while producing transgene-free pollen that cannot establish transgenic weed populations.
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 method effectively reduces transgene escape into non-GM crops and wild species, addressing ecological and trade concerns by ensuring the targeted excision of transgenes from pollen, resulting in improved biocontainment and reduced ecological risks.
Implementation Method 1
a second viable plant containing a genomic DNA, the genomic DNA comprising a DNA encoding a zinc finger nuclease engineered to cleave the genomic DNA at the first and second recognition sequences
Data Source
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AI summary
A method for deleting a region of DNA in a plant. In some embodiments, the method comprises transforming a plant with a nucleic acid molecule, wherein the nucleic acid molecule encodes one or more zinc finger nuclease(s) (ZFNs) operably linked to one or more tissue specific promoter(s), e.g., a pollen specific promoter. Methods include excising native genes in a plant. Accordingly, in some embodiments, ZFNs are engineered that recognize sequences that flank native plant genes. In further embodiments, ZFNs are expressed under the control of developmental stage specific promoters, such that, for example, nucleic acid sequences are specifically excised in plants during relatively late stages of development. Nucleic acid molecules useful for carrying out disclosed methods and plants produced by the methods are included.