Engineered Nucleases for Targeted Plant Genome Mutations
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Solution Overview
Problem
Current methods for reducing saturated fatty acid content in plant oils, such as canola oil, result in random and unspecific genetic changes, making it difficult to achieve desired phenotypic traits in plants modified through mutagenic processes.
Innovation Solution
The use of engineered nucleases to introduce specific mutations in genes such as sec62, KCR2, DGAT1, FAX1, MIR394, and HAD 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 saturated fatty acid content.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional mutagenic processes (chemicals or radiation) are used to reduce saturated fatty acid content, then saturated fatty acid content may be reduced, but random and unspecific genetic changes occur making it difficult to achieve desired phenotypic traits
Solution Approach 1:
The patent replaces traditional mechanical/chemical mutagenesis methods with a molecular-level precision system using engineered nucleases (CRISPR-Cas9, TALENs, ZFNs) that can be programmed to recognize and edit specific DNA sequences. This substitution of the mutagenesis mechanism enables targeted genetic modifications without random changes, directly resolving the contradiction between achieving trait modification and maintaining genetic precision.
Solution Approach 2:
The patent changes the fundamental parameter of genetic modification from random to targeted by using programmable nucleases with specific guide RNA sequences that direct the editing machinery to precise genomic locations. This parameter change in the precision and specificity of genetic modification allows for controlled reduction of saturated fatty acid content while avoiding unwanted genetic changes.
2Adaptability or versatility
If multiple random genetic changes are introduced through mutagenesis, then phenotypic traits may be altered, but it is difficult to determine which specific mutation causes the desired effect
Solution Approach 1:
The patent extracts and isolates the specific genetic locus responsible for saturated fatty acid synthesis (such as FAD2, FAD3, or DGAT genes) and applies targeted nuclease editing only to that specific location. This extraction of the relevant genetic target from the genome allows for precise modification without introducing multiple random changes, making it straightforward to correlate the specific mutation with the desired phenotypic effect.
Solution Approach 2:
The patent introduces guide RNA molecules as intermediaries that mediate between the programmable nuclease system and the target DNA sequence. These guide RNAs provide precise addressing to specific genomic locations, enabling researchers to introduce only the necessary genetic changes and easily track which mutation produces which phenotypic outcome, thereby simplifying the detection and measurement of mutation-phenotype correlations.
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, specifically targeting and reducing stearic acid and total saturated fatty acid levels, providing a more controlled and effective method compared to traditional mutagenic processes.
Implementation Method 1
inducing double stranded DNA breaks at a target site in the plant genome
Implementation Method 2
repaired resulting in a mutation in the genome at the target site, wherein the mutation is a substitution of at least one nucleotide, a deletion of at least one nucleotide, an insertion of at least one nucleotide
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.