Promoter-Activating Sequences for Site-Specific Gene Expression Enhancement
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Solution Overview
Problem
Current technologies lack a generic approach to significantly enhance gene expression using minimal modifications, such as adding ≤ 20bp elements, and existing methods for increasing promoter activity are either inefficient or require transgenic approaches with high regulatory burdens.
Innovation Solution
Introduce promoter activating nucleic acid sequences, configured for site-specific insertion into recipient promoters, using tools like zinc-finger nucleases, CRISPR/Cas systems, or base editors to modify the promoter sequence, thereby increasing expression levels of nucleic acid molecules by up to 40-fold.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If strong promoters or large enhancing elements (e.g., 35S promoter, viral enhancers, introns) are used to increase gene expression, then expression levels are improved, but the size of the genetic modification increases and regulatory complexity increases
Solution Approach 1:
The invention extracts only the essential activating function from large promoter elements, identifying and utilizing small nucleic acid sequences (≤20bp) that contain the core promoter-activating information. This extracts the beneficial effect while removing the bulk of the sequence that contributes to size and complexity
Solution Approach 2:
The invention applies local quality by focusing modification efforts on specific local regions of the promoter (cis-regulatory elements) rather than replacing entire promoters. Small sequences are inserted at precise locations upstream of the transcription start site to activate expression locally without affecting the entire gene structure
2Productivity
If transgenic approaches are used to increase gene expression, then expression levels are improved, but regulatory requirements and complexity increase
Solution Approach 1:
The invention applies partial action by making minimal modifications to the endogenous promoter (adding only ≤20bp sequences) rather than introducing complete transgenic expression cassettes. This partial modification achieves sufficient expression enhancement while avoiding the regulatory burden associated with full transgenic approaches
Solution Approach 2:
The invention enables the endogenous promoter to serve itself by activating its own expression through insertion of small activating sequences. The promoter modifies its own regulatory properties without requiring external transgenic control elements, allowing the organism to regulate the enhanced expression through its native mechanisms
3Ease of manufacture
If minimal modifications (≤20bp elements) are made to promoters, then regulatory complexity is reduced, but expression enhancement is insufficient
Solution Approach 1:
The invention changes critical parameters of the promoter sequence by inserting specific nucleic acid sequences with optimized compositions (rich in A/T or G/C depending on the target promoter type). These parameter changes in sequence composition and structure at key positions produce disproportionate effects on promoter activity, achieving high expression enhancement from minimal sequence changes
Solution Approach 2:
The invention creates composite promoter structures by combining the endogenous promoter sequence with exogenously inserted activating sequences. The resulting hybrid promoter integrates native regulatory elements with optimized activating motifs, achieving enhanced expression that leverages both the natural promoter context and the engineered activating sequences
Data Source
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AI summary
The present invention method for increasing the expression level of a nucleic acid molecule of interest in a cell, preferably a plant cell by means of promoter activating nucleic acid sequences, which are able to increase expression of a nucleic acid molecule of interest in a cell or an organism upon site-specific introduction into a recipient promoter controlling the expression of the nucleic acid molecule of interest. The invention also provides methods to identify such promoter activating elements and methods to introduce them into an organism or a cell to specifically increase the expression of a nucleic acid molecule of interest. Furthermore, the invention also relates to the use of the promoter activating elements to increase the expression of a nucleic acid molecule of interest.