Tissue-Specific Promoters for Precise Plant Gene Editing
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Solution Overview
Problem
Existing methods for gene editing in plant egg cells and embryo tissues are inefficient and lack specificity, particularly in utilizing CRISPR nucleases guided by guide RNAs to target nucleic acid molecules effectively.
Innovation Solution
The use of heterologous promoters, such as egg cell-preferred, embryo tissue-preferred, or meiotic cell-preferred promoters, operably linked with guided nucleases and guide nucleic acids to form a ribonucleoprotein complex that induces targeted modifications, including staggered cuts or transgene insertions in plant genomes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional promoters are used to drive guided nuclease expression, then gene editing can occur, but the specificity and efficiency of editing in egg cells and embryo tissues is low
Solution Approach 1:
The patent applies local quality by using tissue-specific promoters (EA1, ES4 for egg cells; DSUL1, EAL1 for embryo tissues) to drive guided nuclease expression specifically in target tissues rather than constitutively throughout the plant. This ensures high concentrations of the editing machinery are produced precisely where needed, improving both the specificity and efficiency of gene editing in reproductive tissues.
2Productivity
If guided nucleases are expressed without tissue-specific regulation, then general gene editing occurs, but targeted modifications in reproductive tissues are insufficient
Solution Approach 1:
The patent changes the regulatory parameter of promoter specificity to control guided nuclease expression. By switching from constitutive promoters to tissue-specific promoters, the system achieves high productivity (efficient editing) in specific tissues (egg cells, embryo tissues, meiotic cells) while maintaining precision through the spatial restriction of gene expression to these reproductive tissues.
3Reliability
If CRISPR nucleases are used with standard promoters, then gene editing occurs, but the frequency of editing events in reproductive cells is low
Solution Approach 1:
The patent implements local quality by deploying different tissue-specific promoters for different reproductive stages: EA1 and ES4 for egg cell expression, DSUL1 and EAL1 for embryo tissue expression, and meiotic cell-specific promoters for meiotic cell editing. This localized approach increases editing event frequency in each specific cell type while managing complexity through modular promoter selection.
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 enhances the specificity and efficiency of gene editing in plant cells, allowing for precise modifications in egg cells, embryo tissues, or meiotic cells, and facilitates the integration of transgenes into targeted sites.
Implementation Method 1
the at least one guide nucleic acid is capable of forming a complex with the guided nuclease and hybridizing to a target sequence within a genome of the plant
Implementation Method 2
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins guided by guide RNAs to a target nucleic acid molecule, where the nuclease can cleave one or two strands of a target nucleic acid molecule
Data Source
AI summary
This disclosure provides methods and compositions for increasing genome editing and site-directed integration events utilizing guided endonucleases and meiotic cell-preferred, egg cell-preferred or embryo tissue-preferred promoters.