RNA-Guided Nuclease Tomato Genome Editing
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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 plant traits.
Innovation Solution
The use of RNA-guided nucleases such as Cas9 and Cpf1, combined with guide RNAs, to introduce site-specific double-strand breaks in plant genomes, allowing for precise integration of heterologous sequences at desired locations, enabling efficient and targeted modification of plant traits without introducing unwanted variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional Mendelian genetics or recombinant techniques are used for plant breeding and engineering, 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) with a molecular-level system using RNA-guided nucleases (CRISPR-Cas9). This substitution enables precise, targeted genome editing by directing nucleases to specific DNA sequences guided by RNA, achieving both high precision and efficiency in genetic modification without the random variations inherent in traditional methods
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the nuclease enzyme and the target DNA sequence. The guide RNA provides sequence-specific recognition, directing the nuclease to precise genomic locations, thereby enabling targeted modifications without affecting other parts of the genome and avoiding unwanted variations
2Reliability
If traditional plant breeding methods are used, then genetic traits can be modified, but unwanted genetic or epigenetic variations are introduced
Solution Approach 1:
The patent applies local quality by making the genome editing system highly specific to particular genomic locations. The guide RNA is designed to match only the target sequence, ensuring that nuclease activity is confined to the intended locus. This localized action prevents off-target effects and unwanted variations in other parts of the genome, enhancing reliability of targeted modification
Solution Approach 2:
The patent converts the potentially harmful effect of double-strand breaks (which can cause chromosomal rearrangements or mutations) into a beneficial tool for precise editing. By intentionally creating controlled DSBs at specific locations and providing homologous repair templates, the system directs the cell's natural repair mechanisms to produce desired modifications while minimizing unwanted variations
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 genetic modification in plants, allowing for the stacking of preferred alleles and achieving desired traits in tomato plants and seeds with high precision and minimal unintended changes.
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, a type V Cas nuclease, a Cpf1)
Implementation Method 2
The efficiency and reliability of these targeted modification methods are significantly improved relative to traditional plant breeding, and can be used not only to augment traditional breeding techniques but also as a substitute for them
Data Source
AI summary
The disclosure relates to improvements in methods for efficient editing of two or more endogenous tomato genes by insertion of one or more single stranded DNA donor molecules that are not homologous to the insertion sites in the tomato genome.