Engineered Nucleases for Targeted Plant Genome Deletion

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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 desirable oil profiles in modified plants.

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 reductions in saturated fatty acid production by inducing targeted double-stranded DNA breaks and subsequent repairs, resulting in plants with reduced or no activity of these genes.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If chemical or radiation mutagenic processes are used to generate mutants, then mutants with desirable traits can be obtained, but random, multiple and unspecific genetic changes occur

Engineering Contradiction:

Improvespecificity of mutationVSAvoidprecision of mutation location

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The patent replaces chemical/radation mutagenesis with engineered nucleases (molecular scissors) that precisely cut DNA at specific locations. This substitution of mechanical/chemical methods with biological enzymes enables targeted mutations at predetermined genomic sites, eliminating random mutations while achieving the desired phenotypic changes in oil composition.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent introduces engineered nucleases as intermediary molecules that mediate between the researcher's intent and the genomic modification. These nucleases recognize specific DNA sequences and facilitate precise mutations through controlled double-strand breaks, acting as intermediaries that ensure mutations occur only at intended locations rather than randomly throughout the genome.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If multiple random genetic changes are introduced, then desirable phenotypic traits may emerge, but it is difficult to determine which specific mutations caused the phenotype

Engineering Contradiction:

Improvephenotypic diversityVSAvoidinformation about mutation-phenotype relationship

Core Design Contradiction:

Adaptability or versatilityVSLoss of information

Solution Approach 1:

The patent extracts and isolates the specific genetic changes needed to achieve desired phenotypic traits by using engineered nucleases to create targeted mutations only at predetermined locations. This extraction approach eliminates unnecessary random mutations, maintaining phenotypic diversity while preserving complete information about which specific mutations caused which phenotypic changes.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent employs feedback mechanisms where the specific genomic location and nature of mutations are precisely tracked and characterized. By using engineered nucleases with known target sequences, researchers can directly correlate the introduced mutation with the resulting phenotypic change in oil composition, maintaining information about the mutation-phenotype relationship while achieving desired adaptability.

Inventive Principle:

Principle #23Feedback

3Manufacturing precision

If engineered nucleases are used to create specific mutations, then precise control over mutation location is achieved, but the process requires sophisticated molecular biology techniques

Engineering Contradiction:

Improveprecision of mutation locationVSAvoidcomplexity of mutation process

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent employs universal engineered nuclease systems that can be programmed to target any desired genomic location through sequence-specific recognition. This multi-functional approach allows the same basic nuclease platform to create precise mutations at different locations by simply changing the guide sequence, reducing the need for different complex systems for each mutation target while maintaining high precision.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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, providing a specific and controlled method to improve the nutritional profile of plant-derived oils without genetic modification.

Implementation Method 1

inducing double stranded DNA breaks at a target site, wherein said double stranded DNA breaks are induced by the introduction to said cell of a double stranded DNA break inducing enzyme

Methodology Applied

Scientific EffectDouble-stranded DNA break:

Implementation Method 2

wherein said double stranded DNA breaks are repaired resulting in a deletion, substitution or insertion mutation in the genome at said target site

Methodology Applied

Scientific EffectDNA repair:

Data Source

PatentUS11021713B2Engineered nucleases to generate deletion mutants in plants

Publication Date: 2021.06.01 PIONEER HI BREED INTERNATIONAL INC

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.