Universal TALEN Monomer for Multiplexed Gene Editing
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Solution Overview
Problem
Current gene editing technologies, such as TALENs, are limited in their ability to simultaneously target and edit multiple gene sequences with high specificity and efficiency, particularly in plant genomes like those of soybeans, which hampers the precise modification of fatty acid metabolism genes for altering oil composition.
Innovation Solution
The development of a composition comprising a first nucleic acid encoding a transcription activator-like (TAL) effector nuclease monomer capable of binding to a conserved half-site sequence and multiple second nucleic acids encoding TAL effector monomers that bind to different half-site sequences, allowing for the formation of dimers to cleave target genes, specifically targeting genes like FAD3 and FAD2 in soybeans to introduce mutations that alter fatty acid profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a traditional TALEN design process with separate left and right half-TALENs is used, then high specificity for a single target is achieved, but the ability to simultaneously target multiple genes is limited
Solution Approach 1:
The left half-TALEN is designed with a universal FokI nuclease domain that can function with multiple different right half-TALENs. This allows a single left half-TALEN to serve multiple functions by pairing with different right half-TALENs targeting different genes, thereby achieving multi-gene editing capability while maintaining high specificity through conserved half-site recognition
Solution Approach 2:
The TALEN system is divided into modular components: a conserved left half-TALEN with a standardized FokI domain and variable right half-TALENs with different recognition specificities. This segmentation allows independent optimization of each module and facilitates combinatorial pairing to target multiple genes simultaneously
2Manufacturing precision
If multiple separate TALEN constructs are designed for each target gene, then each gene can be targeted with high precision, but the system complexity and number of components increase
Solution Approach 1:
By creating a universal left half-TALEN that can pair with multiple right half-TALENs, the system reduces the total number of constructs needed. Instead of requiring completely separate TALEN pairs for each gene, a single left half-TALEN serves multiple genes, simplifying the overall system while maintaining precision
Solution Approach 2:
The conserved left half-TALEN sequences are merged into a single reusable component that functions across multiple target genes. This merging reduces redundancy and simplifies the number of nucleic acid components that need to be designed, synthesized, and delivered to the cell
Data Source
AI summary
TALEN compositions and methods of use are disclosed, which include using multiplexing compositions to create a targeted mutation in several genes at once, such as the FAD3 A/B/C genes, compositions to create a targeted mutation in a single gene, such as a gene encoding a FAD2 protein, and combinations thereof. The compositions and methods can provide gene-edited plants, plant parts, and plant cells that have improved characteristics compared to the corresponding unaltered plants, plant parts, or plant cells. For example, soybean plants, plant parts and plant cells that are capable of producing a seed with an oil having comparatively higher levels of oleic acid and lower levels of linoleic and linolenic acid than a corresponding seed lacking the targeted mutation are also provided.