FMOS Regulatory Sequence for T1 Seed Editing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current gene editing methods, particularly in plants, face significant challenges in achieving efficient allele replacement, large deletions, and certain base edits due to low specificity and efficiency of DNA modification enzyme systems, leading to high costs and labor intensiveness.
Innovation Solution
The use of a floral mosaic (FMOS) regulatory sequence to mediate the expression of DNA modification enzymes in floral primordia cells and reproductive organs, combined with guide RNAs and donor DNA, to generate a plurality of unique edits in T1 seeds, thereby reducing the number of transformations required.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional gene editing methods are used to achieve allele replacement, large deletions, or certain base edits, then the desired editing outcomes can be obtained, but the process becomes extremely labor intensive and costly requiring generation of one thousand stably transformed events to ensure one allele swap
Solution Approach 1:
The patent applies preliminary action by expressing DNA modification enzymes and guide RNAs specifically in floral primordia cells and reproductive organs before fertilization occurs. This timing allows the enzymes to be present and active during the critical window when genetic material is being transmitted to the next generation, enabling edits to be inherited directly in T1 seeds without requiring multiple transformation cycles
Solution Approach 2:
The patent implements local quality by using tissue-specific promoters to restrict DNA modification enzyme expression exclusively to floral primordia cells and reproductive organs. This localized expression ensures that editing occurs only in the specific tissues where it is needed for heritable changes, while avoiding unnecessary modifications in other plant tissues, thereby increasing editing efficiency and reducing resource waste
2Manufacturing precision
If allele replacement is attempted in plants using homologous recombination, then the desired sequence replacement can be achieved, but the non-homologous end joining pathway is strongly favored making the process very challenging
Solution Approach 1:
The patent applies preliminary action by delivering donor DNA sequences to the cut site before the cell completes repair, ensuring that the homologous recombination pathway has abundant template material available when the repair process initiates. This pre-positioning of donor DNA increases the probability that homologous recombination will be selected over non-homologous end joining
Solution Approach 2:
The patent implements parameter changes by modifying the temporal and spatial parameters of DNA modification enzyme activity. By controlling enzyme expression to occur specifically during floral development stages and in reproductive tissues, the patent creates optimal conditions for homologous recombination to outcompete non-homologous end joining, thereby improving allele replacement efficiency
3Adaptability or versatility
If multiple transformations are performed to generate diverse alleles, then a wide diversity of sequences can be created, but the time and labor required increases significantly
Solution Approach 1:
The patent applies preliminary action by creating multiple allelic variants simultaneously in a single transformation event through the co-expression of multiple guide RNAs and DNA modification enzymes. This approach generates diverse alleles in the T1 generation directly, eliminating the need for sequential transformations and significantly reducing the time and labor required to achieve allelic diversity
Solution Approach 2:
The patent implements universality by designing a single transformation construct that can generate multiple different allelic variants through the coordinated action of multiple guide RNAs targeting different sites or the same site with different outcomes. This multi-functional approach allows one transformation event to accomplish what would traditionally require multiple separate transformations, thereby reducing breeding time and labor
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 approach significantly enhances gene editing efficiency, allowing for the production of multiple unique edits in T1 seeds, including allele replacements, base insertions, deletions, and substitutions, with potentially lower costs and labor compared to traditional methods.
Implementation Method 1
a floral mosaic (FMOS) regulatory sequence, wherein the FMOS regulatory sequence mediates expression of the DNA modification enzyme in at least one of a floral primordia cell and a floral reproductive organ
Data Source
AI summary
Systems and methods for producing a plurality of unique edits in a plant's T1 seed. In one example, a method comprises transforming at least one expression cassette into a plant cell or a plant tissue. The at least one expression cassette may comprise a nucleic acid that encodes a DNA modification enzyme; optionally, a nucleic acid that encodes at least one guide RNA (gRNA); and a floral mosaic (FMOS) regulatory sequence, wherein the FMOS regulatory sequence (i) mediates expression of the DNA modification enzyme in at least one of a floral primordia cell and a floral reproductive organ, and (ii) mediates a plurality of edits in the at least one of the floral primordia and the floral reproductive organ. The method may also include regenerating the plant cell or plant tissue into a T0 plant having a plurality of T1 seed, wherein the T1 seed contain a plurality of unique edits.