Engineered Nucleases for Targeted Plant Genome Mutation
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Solution Overview
Problem
Current methods for modifying plant oil synthesis to reduce saturated fatty acid content in oils, such as canola oil, often result in random and unspecific genetic changes, making it difficult to achieve desired phenotypic traits.
Innovation Solution
The use of engineered nucleases to introduce specific mutations in genes like FatA1, FatA2, Kas2, Kas3, and FatB in Brassica plants, allowing for targeted reduction of saturated fatty acid production by inducing targeted double-stranded DNA breaks and subsequent repair, resulting in plants with reduced or no activity of these genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical or radiation mutagenesis is used to modify plant oil synthesis genes, then saturated fatty acid content can be reduced, but the genetic changes are random and unspecific resulting in multiple unintended mutations
Solution Approach 1:
The mutagenesis process is segmented into targeted steps: designing specific guide RNAs for CRISPR-Cas9 to bind to particular gene sequences (FatA1, FatA2, Kas2, Kas3, or FatB), creating precise double-strand breaks only at desired locations, and allowing controlled repair mechanisms to introduce specific mutations. This segmentation eliminates random mutations across the genome while maintaining focus on oil synthesis genes.
Solution Approach 2:
The patent uses CRISPR-Cas9 as an intermediary system between the researcher and the plant genome. The guide RNA acts as a mediator that directs the Cas9 nuclease to specific target sequences, enabling precise genetic modification without the need for random mutagenesis. This intermediary mechanism ensures that only intended genes are modified while preserving the rest of the genome.
2Measurement precision
If multiple random mutations are introduced to achieve desired phenotype, then saturated fatty acid reduction may occur, but it is difficult to determine which specific mutation caused the phenotypic change
Solution Approach 1:
The patent extracts and isolates the specific genetic changes of interest by using CRISPR-Cas9 to create targeted mutations in only one or a few specific genes (FatA1, FatA2, Kas2, Kas3, or FatB). This extraction approach removes the confounding factor of multiple random mutations, allowing researchers to directly observe and attribute phenotypic changes to the specific engineered mutation without needing to analyze numerous unintended genetic changes.
Solution Approach 2:
The mutagenesis is applied locally to specific genes involved in fatty acid synthesis rather than globally across the entire genome. By targeting only the genes that encode enzymes for saturated fatty acid production, the patent creates localized genetic modifications that can be precisely tracked and correlated with phenotypic outcomes, eliminating the need to analyze genome-wide mutations.
3Manufacturing precision
If engineered nucleases are used to create specific mutations, then the location and effect of mutations can be precisely determined, but the process requires sophisticated gene editing technology
Solution Approach 1:
The CRISPR-Cas9 system provides a universal platform that can target multiple different genes (FatA1, FatA2, Kas2, Kas3, or FatB) using the same core machinery. By designing different guide RNAs with varying sequence specificities, the same Cas9 nuclease can be directed to different genomic locations, enabling precise mutation of any target gene without requiring different nuclease systems for each gene. This universality simplifies the overall process compared to using gene-specific nucleases for each target.
Solution Approach 2:
The patent changes the parameter of guide RNA sequence to redirect the Cas9 nuclease to different target genes. Instead of developing different nucleases for each gene target, the system maintains constant Cas9 and varies only the guide RNA sequence parameters to achieve specificity for different genes involved in fatty acid synthesis. This parameter change approach enables flexible and precise targeting while using a single nuclease platform.
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 enables the production of Brassica plants with significantly reduced saturated fatty acid content in their oils, improving the nutritional profile of the oil without genetic modification, achieving a specific reduction in stearic and palmitic acid levels.
Implementation Method 1
inducing double stranded DNA breaks at target sites
Data Source
AI summary
Methods are provided to mutate, in a targeted manner, the genome of a plant cell using a double stranded DNA break inducing enzyme. Also provided are plants, in particular Brassica plants that yield seeds producing oils having a reduced total saturated fatty acid content, and method for making such plants.