INHT26 Soybean Transgenic Locus Excision via CRISPR
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Solution Overview
Problem
Existing methods for removing selectable marker genes and duplicated transgenes in plant genomes are inefficient and require specific site-specific recombinase systems, which can be complex and difficult to implement.
Innovation Solution
The development of transgenic soybean plant cells with an INHT26 transgenic locus, which includes an originator guide RNA recognition site (OgRRS) and a cognate guide RNA recognition site (CgRRS), allowing for the excision of the transgenic locus using a single guide RNA and an RNA-dependent DNA endonuclease.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If site-specific recombinase systems (e.g., cre-lox) are used to remove selectable marker genes and duplicated transgenes, then the removal can be achieved with high precision, but the system complexity increases and implementation becomes difficult
Solution Approach 1:
The patent replaces the mechanical/protein-based site-specific recombinase system (cre-lox) with a nucleic acid-based CRISPR-Cas system. The CRISPR-Cas system uses guide RNAs and endonucleases to achieve precise excision of transgenes, eliminating the need for complex recombinase proteins and their associated promoter/cassette elements. This substitution dramatically simplifies the system while maintaining or improving precision.
Solution Approach 2:
The patent extracts and removes the complex recombinase system components (cre recombinase, lox sites, promoters, cassettes) from the transgene construct. By designing a minimal transgene structure without these elements and using CRISPR-Cas for removal, the invention eliminates unnecessary system complexity while achieving the desired transgene excision function.
2Ease of manufacture
If non-site specific integration methods are used to insert transgenes, then the integration process is simpler, but the transgene expression levels vary and undesirable rearrangements occur
Solution Approach 1:
The patent applies local quality by creating a specific, defined integration site in the plant genome rather than relying on random insertion. The transgene is designed with specific sequences that enable targeted integration at a predetermined location, ensuring consistent expression levels and avoiding undesirable rearrangements while maintaining the simplicity of the insertion process through CRISPR-Cas mediated integration.
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 efficient excision of the INHT26 transgenic locus, potentially simplifying the removal of undesirable genetic elements and improving the genetic stability of transgenic plants.
Implementation Method 1
an originator guide RNA recognition site (OgRRS) in a first DNA junction polynucleotide of a DAS44406-6 transgenic locus and a cognate guide RNA recognition site (CgRRS) in a second DNA junction polynucleotide of the DAS44406-6 transgenic locus
Implementation Method 2
excision of the transgenic locus using a single guide RNA and an RNA-dependent DNA endonuclease
Data Source
AI summary
Transgenic INHT26 soybean plants comprising modifications of the DAS44406-6 soybean locus which provide for facile excision of the modified DAS44406-6 transgenic locus or portions thereof as well as modifications of the DAS44406-6 soybean locus, methods of making such plants, and use of such plants to facilitate breeding are disclosed.