Rice Genome Editing via CRISPR-Cas9 Allele Stacking
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Solution Overview
Problem
Current plant breeding and engineering methods rely on Mendelian genetics or recombinant techniques, which are inefficient and often introduce unwanted genetic or epigenetic variations, making it difficult to achieve targeted modifications in rice plants.
Innovation Solution
The use of site-specific nucleases like Cas9 and guide RNAs to introduce double-strand breaks in rice plant genomes, allowing for precise integration of altered sequences at desired locations without the need for plasmids or expression vectors, enabling efficient stacking of preferred alleles without introducing unwanted genetic or epigenetic variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional Mendelian genetics or recombinant techniques are used for plant breeding, then genetic modification can be achieved, but efficiency is low and unwanted genetic or epigenetic variations are introduced
Solution Approach 1:
The patent replaces traditional mechanical breeding methods (crossing, selection, and regeneration) with a molecular-level system using CRISPR-Cas9 genome editing. This substitution enables precise targeting of specific genomic loci through guide RNA-directed nucleases, achieving accurate genetic modification without the inefficiencies and unwanted variations associated with conventional breeding approaches
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the CRISPR-Cas9 system and the target genome. The guide RNA provides sequence-specific recognition of target loci, enabling precise genetic modification while avoiding off-target effects and unwanted variations that plague traditional breeding methods
2Adaptability or versatility
If traditional breeding methods are used, then genetic variation can be introduced, but unwanted epigenetic variations and genetic drift occur
Solution Approach 1:
The patent applies local quality by targeting specific genomic loci with CRISPR-Cas9 editing while leaving the rest of the genome unchanged. This localized modification approach introduces desired genetic variations at precise locations without causing unwanted epigenetic variations or genetic drift in other regions, thereby maintaining genetic stability while achieving adaptability
Solution Approach 2:
The patent uses homologous recombination with donor DNA templates that contain desired alleles. By copying precise sequences from donor templates into target loci through homology-directed repair, the method introduces reliable genetic variations without the randomness and unwanted variations inherent in traditional breeding
3Productivity
If multiple alleles are stacked through traditional breeding, then trait enhancement is achieved, but the process is time-consuming and unreliable
Solution Approach 1:
The patent merges multiple CRISPR-Cas9 editing operations into a single transformation event by co-delivering multiple guide RNAs and donor DNA templates. This enables simultaneous stacking of multiple preferred alleles at different genomic loci in one step, dramatically accelerating trait enhancement while ensuring precise integration of each allele at its intended location
Solution Approach 2:
The patent performs preliminary design of guide RNAs and donor DNA templates in silico before transformation. By pre-calculating optimal target sites and designing appropriate donor sequences with homology arms, the method ensures accurate allele stacking at multiple loci simultaneously, improving both speed and precision of trait enhancement
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 method significantly improves the efficiency and reliability of targeted genetic modifications in rice plants, allowing for precise control over gene expression and trait enhancement, reducing unintended changes and enabling the creation of homozygous modified plants in a single step.
Implementation Method 1
Effector molecules for site-specific introduction of a DSB into a genome include various endonucleases (e. g., RNA-guided nucleases such as a type II Cas nuclease, a Cas9...)
Data Source
AI summary
The invention relates to novel plants, seeds and compositions, as well as improvements to plant breeding and methods for creating modifications in plant genomes.