INIR6 Maize Locus Excision for Marker-Free Insect Resistance
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing transgenic plants often contain undesirable rearrangements and selectable marker genes at transgene insertion sites, which are no longer needed once desirable traits are selected, and current methods for removing these require site-specific recombinase systems that are cumbersome.
Innovation Solution
The introduction of an INIR6 transgenic locus in maize plants, comprising an originator guide RNA recognition site (OgRRS) and a cognate guide RNA recognition site (CgRRS), allows for the excision of the transgenic locus using an RNA-dependent DNA endonuclease and guide RNA, enabling efficient removal of unwanted transgenic elements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of substance
If site-specific recombinase systems (e.g., cre-lox) are used to remove selectable marker genes and duplicated transgenes, then the marker genes and duplications can be removed, but the process becomes cumbersome and complex
Solution Approach 1:
The patent extracts and removes the selectable marker gene and duplicated transgene sequences from the transgenic locus through precise genomic editing, leaving only the desired transgene intact. This directly addresses the contradiction by eliminating the unwanted genetic material without requiring complex recombinase systems.
Solution Approach 2:
The invention uses PCR amplification to generate the desired transgene sequence independently, creating a clean copy that can be reintegrated into the genome without the marker genes or duplications. This copying approach simplifies the process compared to using recombinase systems to remove unwanted elements.
2Reliability
If transgenes are inserted into the plant genome, then desirable traits (insect resistance, herbicide tolerance) are achieved, but undesirable rearrangements and marker genes are introduced at the insertion site
Solution Approach 1:
The patent removes the harmful marker genes and rearranged sequences from the transgenic locus while preserving the functional transgene. This extraction process eliminates the harmful factors generated by initial transgene insertion while maintaining the desirable trait expression.
Solution Approach 2:
The invention discards the selectable marker genes and duplicated sequences that were temporarily necessary for transformation, then recovers and preserves only the essential transgene sequence for trait conferment. This allows the plant to retain desirable traits without the harmful accompanying elements.
3Ease of manufacture
If selectable marker genes are included in transgenic constructs, then selection of transformed plants is facilitated, but the marker genes remain in the final transgenic event and are no longer needed
Solution Approach 1:
The patent performs the removal of marker genes as a preliminary step before final transgenic event characterization. By eliminating the marker genes early in the process, the final transgenic plants contain only the necessary trait-conferring sequences without unwanted genetic elements.
Solution Approach 2:
The invention extracts and removes the selectable marker gene sequences from the transgenic construct after transformation but before final deployment. This allows the marker genes to serve their selection purpose temporarily, then be eliminated from the final transgenic product.
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 method enables the targeted and efficient removal of transgenic loci, preserving desirable traits while eliminating unnecessary markers and rearrangements, facilitating the recovery of germplasm tailored for specific geographic locations and grower preferences.
Implementation Method 1
contacting the INIR6 transgenic locus with: (i) an RNA dependent DNA endonuclease; and (ii) a guide RNA capable of hybridizing to the guide RNA hybridization site of the originator guide RNA recognition site and the cognate guide RNA recognition site; wherein the RNA dependent DNA endonuclease recognizes and binds to the guide RNA-originator guide RNA recognition site and cognate guide RNA-cognate guide RNA recognition site complexes and cleaves the DNA at or near the guide RNA hybridization sites
Data Source
AI summary
Transgenic INIR6 maize plants comprising modifications of the DP-4114 maize locus which provide for facile excision of the modified DP-4114 transgenic locus or portions thereof, methods of making such plants, and use of such plants to facilitate breeding are disclosed.