INIR6 Maize Locus Excision for Marker-Free Trait Breeding
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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 conferred, and current methods for removing these require site-specific recombinase systems.
Innovation Solution
The introduction of an INIR6 transgenic locus with an originator guide RNA recognition site (OgRRS) in a first DNA junction polynucleotide and a cognate guide RNA recognition site (CgRRS) in a second DNA junction polynucleotide, allowing for the excision of the transgenic locus using an RNA-dependent DNA endonuclease and guide RNA, enabling targeted removal of transgenic loci.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If site-specific recombinase systems (e.g., cre-lox) are used to remove selectable marker genes and duplicated transgenes, then the transgenic locus can be cleaned up, but the method requires incorporation of recombination site sequences at particular locations within the transgene, increasing device complexity
Solution Approach 1:
The patent extracts and removes the selectable marker gene and duplicated transgene sequences from the transgenic locus using RNA-dependent DNA endonuclease and guide RNA targeting, leaving only the essential transgenic trait sequences. This extraction approach cleans up the locus without requiring permanent incorporation of recombination site sequences.
Solution Approach 2:
The patent introduces an intermediary RNA-dependent DNA endonuclease system with guide RNA that temporarily targets and cleaves specific sequences at the transgenic locus. This intermediary mechanism enables precise removal of unwanted sequences without permanently modifying the locus structure with recombination sites.
2Ease of manufacture
If transgenes are placed into different positions in the plant genome through non-site specific integration, then insertion is simpler, but the transgenes can exhibit different levels of expression and contain undesirable rearrangements
Solution Approach 1:
The patent employs a self-targeting mechanism where the RNA-dependent DNA endonuclease with guide RNA automatically recognizes and binds to specific sequences at the transgenic locus. This self-service approach ensures consistent targeting and removal regardless of the initial insertion position, standardizing the cleanup process across different integration sites.
3Ease of operation
If selectable marker genes are retained in transgene insertion sites, then selection of transgenic plants is easier, but the marker genes are no longer needed once desirable traits are conferred and occupy unnecessary genomic space
Solution Approach 1:
The patent performs preliminary removal of the selectable marker gene and duplicated sequences during the transgenic locus construction process, before the plants need to be propagated. The RNA-dependent DNA endonuclease system is applied in advance to clean up the locus, eliminating the need for marker genes in the final transgenic lines.
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 allows for the efficient excision of transgenic loci, facilitating the recovery of germplasm with tailored traits and removing unnecessary transgenic traits without disrupting other loci, enhancing breeding efficiency and trait specificity.
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 OgRRS and the CgRRS
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.