Optimized Plant Promoter Sequences for Editing Efficiency

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Solution Overview

Problem

Current methods for expressing polynucleotides of interest in plants, such as editing reagents like nucleases and guide RNAs, face inefficiencies in expression and heritable mutation introduction, requiring optimization of upstream regulatory elements to enhance expression levels and editing efficiency.

Innovation Solution

The use of nucleic acid molecules with optimized promoter sequences, including synthetic motif sequences and linkers, to drive high expression levels and favorable patterns of polynucleotides, specifically incorporating sequences with high sequence identity to certain SEQ ID NOs, and introducing mutations at specific positions to enhance transcription initiation function.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If conventional promoters are used to express editing reagents in plants, then the expression system is simple, but the expression level and editing efficiency are insufficient

Engineering Contradiction:

Improveediting efficiencyVSAvoidpromoter structure complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The promoter is divided into multiple functional modules including core promoter elements, upstream activating sequences, and synthetic motif sequences. Each module can be independently optimized and combined to achieve high expression levels while maintaining systematic organization.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The promoter construct combines natural plant promoter sequences with synthetic motif sequences to create a composite regulatory element that leverages both the biological compatibility of natural sequences and the enhanced functionality of synthetic elements designed for high expression.

Inventive Principle:

Principle #40Composite materials

2Productivity

If upstream regulatory elements are optimized to enhance expression, then editing efficiency improves, but the design and implementation complexity increases

Engineering Contradiction:

Improveheritable edit introduction efficiencyVSAvoidpromoter optimization complexity

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The promoter sequence parameters are systematically optimized by modifying specific nucleotide positions, adjusting motif sequence compositions, and varying regulatory element configurations to achieve enhanced expression while following established optimization protocols.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The promoter sequences are pre-optimized and characterized before use in editing applications. Synthetic motif sequences are designed and validated in advance, allowing researchers to directly implement high-performance promoters without performing complex optimization experiments during the editing process.

Inventive Principle:

Principle #10Preliminary action

3Productivity

If expression levels of editing reagents are increased, then heritable mutation frequency increases, but off-target effects may increase

Engineering Contradiction:

Improvemutation introduction frequencyVSAvoidediting precision

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The promoter enables controlled, stage-specific expression of editing reagents during plant development. Expression is activated at optimal time points and tissue types, ensuring sufficient editing efficiency while limiting prolonged exposure that could increase off-target effects.

Inventive Principle:

Principle #19Periodic action

Solution Approach 2:

The promoter is designed to drive expression specifically in target tissues or developmental stages where editing is most effective. This spatial and temporal specificity ensures high local expression levels at the editing site while maintaining lower overall expression levels that reduce the risk of off-target effects in other plant tissues.

Inventive Principle:

Principle #3Local quality

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 approach significantly increases the expression and editing frequency of editing reagents in plants, improving the efficiency of introducing heritable edits and mutation outcomes, such as homology-directed repair, compared to control promoters.

Implementation Method 1

the promoter sequence has transcription initiation function

Methodology Applied

Scientific EffectTranscription initiation:

Data Source

PatentUS20250011801A1Promoter elements for improved polynucleotide expression in plants

Publication Date: 2025.01.09 CONFLUENCE GENETICS LLC

AI summary

Provided herein are compositions and methods for expressing a polynucleotide of interest in a plant or plant part. Compositions include nucleic acid molecules comprising a promoter sequence, and DNA constructs comprising the promoter molecule operably linked to one or more polynucleotides of interest. Plants and plant parts comprising the compositions or regenerated according to the methods are also provided.