CRISPR Plant Genome Editing for Precise Multi-Locus Modification
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Solution Overview
Problem
Traditional plant breeding methods face limitations in efficiently and reliably introducing targeted genetic modifications without introducing unwanted genetic or epigenetic variations, particularly in achieving homozygous modifications across multiple loci simultaneously.
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 through non-homologous end joining, enabling the stacking of preferred alleles without unwanted genetic variation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional plant breeding methods are used to introduce targeted genetic modifications, then genetic modifications can be achieved, but extensive crossing 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 targeting of specific genomic loci through complementary base pairing between guide RNA and DNA, eliminating the need for extensive crossing and the associated unwanted genetic variation.
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 contains a spacer sequence complementary to the target locus, enabling specific recognition and binding, which directs the nuclease to the exact location for modification without affecting other genomic regions.
2Productivity
If traditional breeding methods are used to achieve homozygous modifications across multiple loci, then genetic modifications can be obtained, but the process requires multiple generations and extensive crossing
Solution Approach 1:
The patent performs preliminary action by introducing the RNA-guided nuclease system and multiple guide RNAs targeting different loci simultaneously into the plant cell. This allows all desired genomic modifications to be established in a single generation, eliminating the need for sequential crossing and selection across multiple generations that would otherwise be required to achieve homozygous modifications at multiple loci.
3Manufacturing precision
If RNA-guided nucleases are used for targeted genome editing, then precision and efficiency of modification are improved, but the complexity of the editing system increases
Solution Approach 1:
The patent segments the genome editing function into distinct modular components: (1) the nuclease enzyme (Cas9 or Cpf1) that performs the cutting, (2) the guide RNA molecules that provide target specificity through their spacer sequences, and (3) the PAM sequence recognition requirement. This segmentation allows each component to be independently optimized and combined, achieving high precision while maintaining systematic organization.
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 significantly enhances the efficiency and reliability of targeted genome editing in plants, allowing for simultaneous modification of multiple loci and achieving homozygous modifications in a single step, reducing the need for extensive crossing and minimizing unintended genetic changes.
Implementation Method 1
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
Implementation Method 2
allowing for precise integration of heterologous sequences at desired locations through non-homologous end joining
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.