Transposable Element System for Stable Genome Integration
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Solution Overview
Problem
Current transposable element technologies for genetic transformation in insects are inefficient and unstable, as they often leave behind transposon ends that can lead to unwanted excisions and mobilization across species barriers, posing environmental risks and destabilizing genetic insertions.
Innovation Solution
A transposable element system using at least four inverted repeats, where the DNA of interest is inserted between pairs of opposing repeats, allowing excision by a transposase to leave the DNA integrated without flanking transposon-derived repeats, utilizing minimal repeats and recombinase-mediated inversion to ensure stability and control over transposition and excision steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If transposable elements with inverted repeats are used for genetic transformation, then transformation efficiency is improved, but the inserted DNA becomes unstable and susceptible to excision by transposase
Solution Approach 1:
The invention extracts and removes the transposon ends (inverted repeats) from the inserted DNA construct after transformation. By using a transposase enzyme to excise the transposon flanking sequences, the patent leaves behind only the desired DNA insert without the mobile elements, thereby eliminating the instability caused by residual transposon activity while maintaining the transformation efficiency benefits of the original transposon-based delivery system
Solution Approach 2:
The invention segments the transposable element system into distinct functional components: (1) the transposon flanked by inverted repeats for efficient transformation, (2) the transposase enzyme for controlled excision, and (3) the desired DNA insert. This segmentation allows the system to first function as a complete transposon for high-efficiency integration, then be processed to remove only the necessary flanking sequences, leaving a stable insert without compromising the initial transformation efficiency
2Ease of manufacture
If transposon ends are left flanking the inserted DNA, then transformation is simplified, but environmental mobilization risk increases
Solution Approach 1:
The invention converts the potentially harmful presence of transposon ends into a beneficial process by utilizing the transposase enzyme's natural excision activity. The same transposase that could cause unwanted mobilization is instead harnessed to precisely remove the transposon flanking sequences, transforming the risk of environmental mobilization into a controlled mechanism for creating stable, safe genetic inserts devoid of mobile elements
3Productivity
If transposase is present to enable transposition, then transformation efficiency is improved, but insertion stability decreases due to potential excision
Solution Approach 1:
The invention applies preliminary action by first introducing the complete transposon construct with inverted repeats to achieve efficient transformation, then subsequently applying transposase to excise the flanking sequences. This two-step preliminary action ensures that transformation efficiency is maximized in the first phase, followed by stabilization in the second phase, thereby resolving the contradiction between efficiency and stability
Data Source
AI summary
There is provided a transposable element comprising at least four inverted repeats, at least two of which are each inverted in relation to another, the element comprising DNA for insertion into a host genome, the DNA being located between two pairs of opposing repeats excisable by a transposase in situ to leave said DNA without flanking transposon-derived repeats in the host genome. Also provided is a transposable element comprising at least three inverted repeats, at least one of which is inverted in relation to the others, wherein at least one non-terminal repeat is a minimal repeat. Both these elements allow for greater efficiency of insertion of nucleotide sequences into the genome.