Antisense RNA Allele for Corn Lodging Resistance

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Solution Overview

Problem

There is a challenge in achieving increased yield and resistance to lodging in corn crops through manipulation of the gibberellin pathway, which has been successful in other cereal crops but remains elusive in corn.

Innovation Solution

A modified corn plant with a mutant allele of the endogenous GA20 oxidase_5 locus is created through targeted genome editing, involving deletions in specific regions of the GA20 oxidase_5 and Zm.SAMT genes to produce an antisense RNA molecule that suppresses the expression of the GA20 oxidase_5 gene, thereby regulating gibberellin levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If GA pathway manipulation is applied to corn crops, then yield and lodging resistance can be improved, but the technical challenge remains unsolved compared to other cereal crops

Engineering Contradiction:

ImproveyieldVSAvoidsuccess rate of GA pathway manipulation

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

Instead of directly manipulating GA biosynthesis genes to increase GA levels, the invention uses antisense RNA to suppress GA20 oxidase_5 gene expression, thereby reducing GA levels to achieve semi-dwarf phenotype and lodging resistance. This inverse approach of suppressing rather than activating the pathway proves effective in corn where direct activation had failed.

Inventive Principle:

Principle #13The other way round (Inversion)

Solution Approach 2:

The invention introduces an intermediary mechanism (antisense RNA molecule) between the genome editing tool and the target gene. The antisense RNA acts as a mediator that specifically binds to and suppresses the sense mRNA of GA20 oxidase_5, providing precise control over gene expression without directly modifying the coding sequence.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If genome editing is used to create mutant alleles, then precise control of GA levels is achieved, but complex genomic modifications are required

Engineering Contradiction:

Improveprecision of GA level regulationVSAvoidcomplexity of genomic modification

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The invention extracts only the essential functional element needed for antisense RNA production by deleting specific regions (transcription termination sequence of Zm.SAMT gene and/or intergenic region) while leaving the rest of the genome intact. This minimal modification approach achieves precise GA level control without requiring complex multi-gene edits.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention changes the transcriptional parameters of the Zm.SAMT gene by modifying its termination sequence and intergenic region, which alters the expression pattern to produce antisense RNA. This parameter change (from normal transcription to antisense transcription) provides precise control over GA20 oxidase_5 gene expression.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If transcription termination sequence is deleted to produce antisense RNA, then GA levels are effectively regulated, but genomic structure is altered

Engineering Contradiction:

Improveyield improvementVSAvoidgenomic structure stability

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The invention performs preliminary genomic modification by deleting the transcription termination sequence and/or intergenic region before the antisense RNA production begins. This pre-established genomic structure ensures that antisense RNA is constitutively produced, leading to stable and continuous regulation of GA levels throughout plant development.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention merges the Zm.SAMT gene promoter with the antisense sequence by deleting the termination sequence, creating a fused transcriptional unit. This merging allows the Zm.SAMT promoter to drive antisense RNA production, combining regulatory elements from one gene with the antisense function to achieve stable genomic integration of the trait.

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

The modified corn plants exhibit improved yield and lodging resistance by effectively regulating gibberellin levels, resulting in enhanced plant growth and stability.

Implementation Method 1

the mutant allele produces a RNA molecule comprising an antisense sequence complementary to all or part of the sense strand of the endogenous GA20 oxidase_5 gene

Methodology Applied

Scientific EffectComplementary base pairing:

Data Source

PatentUS20220195450A1Methods and compositions for generating dominant short stature alleles using genome editing

Publication Date: 2022.06.23 MONSANTO TECHNOLOGY LLC

AI summary

The present disclosure provides compositions and methods for altering gibberellin (GA) content in corn or other cereal plants. Methods and compositions are also provided for altering the expression of genes related to gibberellin biosynthesis through editing of a specific GA20 oxidase gene or locus to produce a genomic deletion or disruption that brings an antisense sequence of the GA20 oxidase gene under the control of a neighboring SAMT gene promoter. Modified plant cells and plants having a dominant allele reducing the expression or activity of one or more GA oxidase genes are further provided comprising reduced gibberellin levels and improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types.