Stabilizing dsRNA Expression Constructs in Transgenic Plants
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Solution Overview
Problem
Current methods for stable expression of RNAi constructs in plants face challenges in optimizing the production, stabilization, and uptake of dsRNAs for effective pest control, while avoiding transgene silencing in the host plant.
Innovation Solution
The development of nucleic acid segments and expression constructs that include specific siRNA sequences, introns, and spacer sequences to enhance the stability and specificity of dsRNA production, allowing for targeted gene suppression in plant pests and pathogens, and the use of recombinant DNA constructs to express these sequences in transgenic plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If dsRNA is produced from a transgene in plants, then gene suppression in target pests can be achieved, but transgene silencing and cleavage of transgene transcript occur reducing dsRNA production
Solution Approach 1:
The transgene is divided into multiple segments including sense sequence, antisense sequence, and spacer sequence. This segmentation prevents the plant's silencing machinery from recognizing the entire construct as foreign, thereby reducing transgene silencing while maintaining dsRNA production capability.
Solution Approach 2:
A spacer sequence acts as an intermediary element between the sense and antisense sequences. This spacer prevents direct hybridization of the transgene transcript with endogenous plant RNAs, reducing cleavage events and stabilizing transgene expression while still allowing dsRNA formation for pest control.
2Productivity
If dsRNA length is increased to improve uptake by pests, then gene suppression efficacy improves, but transgene silencing and transcript cleavage increase
Solution Approach 1:
Different regions of the dsRNA construct have different functions: the sense and antisense sequences (local regions) are designed for high similarity to target pest genes to ensure specific gene suppression, while the spacer sequence has low similarity to plant genes to prevent silencing. This local quality differentiation optimizes both efficacy and stability.
Solution Approach 2:
The construct parameters are optimized by adjusting the length and sequence composition of different regions. The sense and antisense sequences are designed with high complementarity to target pest sequences, while the spacer sequence parameters are adjusted to minimize homology with plant sequences, thereby balancing uptake efficiency with transgene stability.
3Measurement precision
If high similarity between dsRNA and target gene transcript is used to improve specificity, then gene suppression precision improves, but transgene silencing in host plant increases
Solution Approach 1:
The construct exhibits asymmetry in sequence similarity: high similarity between sense/antisense sequences and target pest gene (for specificity), but low similarity between spacer sequence and plant genes (to avoid silencing). This asymmetric design allows specific pest targeting while maintaining transgene stability in the host plant.
Solution Approach 2:
Instead of designing the entire transgene to be similar to the target (which would cause silencing), the invention inverts the approach by making only the functional regions (sense and antisense sequences) similar to the target, while the spacer region is deliberately made dissimilar to plant sequences, thereby achieving specificity without triggering silencing.
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 enables efficient and specific suppression of target gene expression in plant pests, reducing transgene silencing and improving the efficacy of pest control by optimizing dsRNA production and uptake, leading to effective phenotypic effects such as mortality or inhibition of feeding in target organisms.
Implementation Method 1
The dsRNA may be cleaved by enzymes known as dimeric RNase III ribonucleases (also called 'dicer' enzymes) into segments approximately 21-25 base pairs in length
Implementation Method 2
The siRNA causes specific RNAse activity in a RNA-induced silencing complex ('RISC') to hydrolyze the target gene mRNA, thereby post-transcriptionally suppressing expression of the target gene
Implementation Method 3
dsRNA present in plants may also guide DNA methylation of targeted chromatin regions, resulting in gene silencing
Data Source
AI summary
The invention provides methods for selecting nucleotide sequences that yield dsRNA-mediated gene suppression in a target organism and enable their uptake by the target organism. The invention further provides expression constructs that confer stabilized expression of such sequences in a transgenic host cell, and methods for their use. Also provided are organisms, cells and tissues prepared by a method of the invention.