INIR20 Transgenic Soybean Junction Deletions for Precise Locus Excision
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Solution Overview
Problem
Existing transgenic plants often have undesirable rearrangements and marker genes at insertion sites, requiring complex methods for removal and integration of new genes, which can disrupt normal Mendelian transmission and expression of desired traits.
Innovation Solution
The introduction of 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 of the MON87701 transgenic locus, allowing for the excision of the INIR20 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
1Ease of manufacture
If non-site specific integration methods are used to place transgenes into the plant genome, then transgene insertion is achieved, but undesirable rearrangements and marker genes are introduced at insertion sites
Solution Approach 1:
The patent extracts and removes unwanted elements (marker genes, duplicated transgenes, and rearranged DNA) from the transgene insertion site through targeted excision methods, leaving only the desired transgene expression functionality while eliminating the harmful rearrangements and unnecessary marker genes that were introduced during initial integration
Solution Approach 2:
The patent incorporates site-specific recombination sites (loxP, FRT, or LAC operators) and selectable marker genes during the initial transgene integration step, preparing the insertion site for future targeted removal of unwanted elements while maintaining the desired transgene expression, thereby preventing long-term genetic disruption
2Reliability
If site-specific recombination systems are used to remove marker genes and duplicated transgenes, then genetic integrity is improved, but complex recombination site sequences must be incorporated at particular locations
Solution Approach 1:
The patent employs universal recombination site sequences (loxP, FRT, or LAC operators) that can be integrated into multiple different transgene constructs and plant genomes using the same mechanism, allowing standardized removal of marker genes and duplicated transgenes across various transgenic events without requiring construct-specific recombination sites
Solution Approach 2:
The patent uses selectable marker genes as intermediary elements that facilitate the initial integration process and can be subsequently removed through site-specific recombination, serving as temporary placeholders that enable transgene insertion while being easily eliminated later to achieve clean genetic architecture
3Reliability
If multiple independent insertions of transgenes are made at specific locations, then desired traits are achieved, but Mendelian transmission may be disrupted
Solution Approach 1:
The patent separates the transgene expression function from the genetic transmission function by using site-specific recombination sites that allow independent control: the transgene can be expressed from the insertion site while the recombination sites enable clean removal of marker genes without affecting the inheritance of the desired transgene trait, thereby maintaining Mendelian transmission ratios
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 precise excision of transgenic loci, maintaining desired traits while minimizing genetic disruption, facilitating the integration of new genes and improving breeding strategies for transgenic plants.
Implementation Method 1
excision of the INIR20 transgenic locus using an RNA-dependent DNA endonuclease and guide RNA
Data Source
AI summary
Transgenic INIR20 soybean plants comprising modifications of the MON87701 soybean locus which provide for facile excision of the modified MON87701 transgenic locus or portions thereof, methods of making such plants, and use of such plants to facilitate breeding are disclosed.