INIR6 Transgenic Maize Locus Excision for Precise Marker Removal

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Solution Overview

Problem

Existing transgenic plants face issues with non-specific integration of transgenes leading to undesirable rearrangements and the presence of unnecessary marker genes, which complicates breeding and trait expression.

Innovation Solution

Incorporation of an originator guide RNA recognition site (OgRRS) and a cognate guide RNA recognition site (CgRRS) in the transgenic locus, allowing for the use of an RNA-dependent DNA endonuclease to excise the transgenic locus precisely, using guide RNA and RdDe to target and remove the unwanted DNA segments.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If non-site specific integration methods are used to insert transgenes, then the transformation process is simpler and more efficient, but undesirable rearrangements and unexpected expression patterns occur

Engineering Contradiction:

Improvetransformation process simplicityVSAvoidtransgene integration precision

Core Design Contradiction:

Ease of manufactureVSManufacturing precision

Solution Approach 1:

The transgene construct is divided into separate functional modules: the transgene of interest, selectable marker genes, and site-specific recombination sequences (loxP sites). This segmentation allows the transgene to be inserted non-specifically for ease of transformation, while the recombination sequences enable precise removal later without affecting the transgene itself.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Site-specific recombination sequences (loxP sites) are incorporated into the transgene construct during the transformation process. These sequences are prepared in advance at specific locations within the transgene, enabling future precise removal of marker genes while preserving the transgene of interest, thus resolving the precision issue without complicating the initial transformation.

Inventive Principle:

Principle #10Preliminary action

2Reliability

If selectable marker genes are included in transgene insertion sites, then transgene expression and trait conferment can be selected for, but unnecessary genetic elements remain that complicate breeding

Engineering Contradiction:

Improvetransgene expression reliabilityVSAvoidgenetic architecture complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The selectable marker genes are positioned between loxP sites within the transgene insertion site. After the transgene has served its purpose for selection and trait conferment, the marker genes can be precisely extracted (removed) through site-specific recombination, leaving only the desired transgene in the plant genome. This extraction process simplifies the genetic architecture for breeding while maintaining selection reliability during the transformation process.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The loxP sites act as intermediary elements that enable the controlled removal of selectable marker genes. These recombination sequences serve as mediators between the transgene and the selectable markers, allowing precise excision of the markers without affecting the transgene of interest. This intermediary mechanism resolves the contradiction by enabling both reliable selection during transformation and simplified genetics for breeding.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Adaptability or versatility

If multiple independent transgene insertions are made to achieve desired traits, then trait expression can be optimized, but genetic stability and Mendelian transmission are compromised

Engineering Contradiction:

Improvetrait expression versatilityVSAvoidgenetic stability

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

Multiple transgenes that would traditionally be inserted at separate locations are merged into a single insertion site, with each transgene flanked by its own loxP sites. This merging approach maintains the versatility of expressing multiple traits while ensuring genetic stability, as all transgenes are removed together through a single recombination event and maintain proper Mendelian inheritance patterns.

Inventive Principle:

Principle #5Merging (Combining)

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

Facilitates the efficient removal of transgenic loci and marker genes, enabling tailored trait combinations and breeding strategies without disrupting other genetic elements, enhancing genetic stability and breeding efficiency.

Implementation Method 1

use of an RNA-dependent DNA endonuclease (RdDe) and a guide RNA (gRNA) to excise the transgenic locus

Methodology Applied

Scientific EffectRNA-dependent DNA endonuclease activity: Enzyme

Implementation Method 2

an originator guide RNA recognition site (OgRRS) in a first DNA junction polynucleotide of a DP-4114 transgenic locus and a cognate guide RNA recognition site (CgRRS) in a second DNA junction polynucleotide of the DP-4114 transgenic locus

Methodology Applied

Scientific EffectGuide RNA recognition:

Data Source

PatentUS20250243497A1INIR6 transgenic maize

Publication Date: 2025.07.31 INARI AGRICULTURE TECHNOLOGY INC

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.