Guide RNA/Cas Plant Genome Editing for Precise Targeted Insertion
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Solution Overview
Problem
Existing methods for altering plant genomes are inefficient and unpredictable, often resulting in random integration of transgenes and lack the ability to produce fertile plants with defined genetic modifications.
Innovation Solution
Employing a guide RNA/Cas endonuclease system to introduce targeted double-strand breaks in plant genomes, allowing for precise editing and insertion of specific polynucleotides, and utilizing breeding methods with a two-component RNA guide and Cas endonuclease system to select plants with desired genetic alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Agrobacterium infection or biolistic particle bombardment is used for plant transformation, then foreign DNA sequences can be inserted into the plant genome, but the integration is random and unpredictable in copy number
Solution Approach 1:
The patent employs site-specific recombination systems (such as Cre-loxP, Flp-FRT, or attB-attP) that require preliminary arrangement of specific recognition sequences at the target genomic location. The transgene is constructed with matching recombination sites, ensuring that integration occurs only at predetermined locations rather than randomly throughout the genome.
Solution Approach 2:
The patent introduces intermediary recombination sites (loxP, FRT, att sites) as mediators between the transgene and the target genome. These intermediary sequences serve as docking stations that facilitate precise integration through site-specific recombination enzymes, eliminating the randomness of conventional transformation methods.
2Manufacturing precision
If homologous DNA recombination is used to target DNA sequences, then site-specific modification is achieved, but the process requires complex DNA constructs with flanking homologous sequences
Solution Approach 1:
The patent extracts and utilizes only the essential functional elements for site-specific recombination (the recombination sites and enzyme) while eliminating the need for extensive flanking homologous sequences required by traditional homologous recombination. This simplifies the DNA construct while maintaining precise targeting capability.
Solution Approach 2:
The patent employs universal recombination systems (Cre-loxP, Flp-FRT) that can be applied to any target genome sequence by simply placing the appropriate recombination sites at the desired location. This universal approach replaces the need for custom-designed homologous flanking sequences for each targeting event, reducing construct complexity.
3Productivity
If traditional transformation methods are used, then plants can be transformed with transgenes, but the copy number and integration pattern are unpredictable
Solution Approach 1:
The patent changes the fundamental parameter of integration mechanism from random insertion to site-specific recombination. By controlling the location of recombination sites in the genome and the expression of recombination enzymes, the integration pattern becomes predictable while maintaining high transformation efficiency through the specificity and efficiency of the recombination system.
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
Enables efficient and precise modification of plant genomes, facilitating the production of fertile plants with defined genetic changes and enabling the insertion or deletion of polynucleotides at targeted sites.
Implementation Method 1
artificially induced site-specific genomic double-stranded breaks in plant cells were repaired by homologous recombination with exogenously supplied DNA
Data Source
AI summary
Compositions and methods are provided for genome modification of a target sequence in the genome of a plant or plant cell. The methods and compositions employ a guide RNA/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a plant, plant cell or seed. Also provided are compositions and methods employing a guide polynucleotide/Cas endonuclease system for genome modification of a nucleotide sequence in the genome of a cell or organism, for gene editing, and/or for inserting or deleting a polynucleotide of interest into or from the genome of a cell or organism. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Breeding methods and methods for selecting plants utilizing a two component RNA guide and Cas endonuclease system are also disclosed. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell.