Plant Protoplast Genome Editing via Cas9 RNP Delivery
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Solution Overview
Problem
The widespread use of genome-edited plants is hindered by regulatory concerns regarding genetically modified organisms (GMOs), as existing methods often introduce foreign DNA sequences into plant genomes, and there is a lack of efficient methods for genome editing in plants using CRISPR/Cas systems.
Innovation Solution
Introducing a Cas protein and a guide RNA directly into isolated plant protoplasts to edit the genome, thereby regenerating genome-edited plants without inserting foreign DNA, utilizing preassembled Cas9 protein-gRNA ribonucleoproteins (RNPs) to minimize off-target effects and increase editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If non-integrating plasmids encoding programmable nucleases are transfected into plant cells, then genome editing can be achieved, but foreign DNA sequences are inserted into the host genome causing regulatory concerns
Solution Approach 1:
The patent extracts and separates the Cas9 protein from its encoding DNA, delivering only the protein-gRNA ribonucleoprotein complex into plant cells. This eliminates the need for plasmid transfection and prevents foreign DNA integration into the host genome, resolving the contradiction between achieving genome editing and avoiding foreign DNA insertion
Solution Approach 2:
The patent uses preassembled Cas9 protein-gRNA ribonucleoproteins as an intermediary delivery system. This RNP complex serves as a mediator that performs genome editing functions without requiring DNA integration, allowing precise genome modification while avoiding the harmful effect of foreign DNA insertion
2Ease of manufacture
If Agrobacterium is used for genome editing, then programmable nucleases can be delivered, but foreign DNA sequences including nuclease genes remain in the plant genome
Solution Approach 1:
The patent extracts the Cas9 protein function from the Agrobacterium-mediated DNA transfer system. By delivering preassembled RNPs directly rather than using Agrobacterium to transfer DNA, the method eliminates the insertion of foreign DNA sequences including T-DNA and nuclease genes into the plant genome
Solution Approach 2:
The patent employs transient delivery of Cas9 protein-gRNA RNPs that function temporarily and are then degraded by cellular proteases. This disposable approach allows genome editing to occur without permanent integration of foreign DNA, contrasting with the persistent foreign DNA insertion caused by Agrobacterium
3Manufacturing precision
If plasmids are transfected into plant protoplasts, then genome editing can occur, but plasmids are degraded by endogenous nucleases causing DNA fragment insertion at target sites
Solution Approach 1:
The patent extracts the genome editing function from plasmid DNA by delivering preassembled Cas9 protein-gRNA ribonucleoproteins. This eliminates the need for plasmid transfection and prevents the degradation of plasmid DNA by endogenous nucleases, thereby avoiding the harmful insertion of DNA fragments at target sites
Solution Approach 2:
The patent performs preliminary assembly of the Cas9 protein and gRNA into functional ribonucleoprotein complexes before delivery into plant cells. This preassembly ensures that the editing machinery is ready to function immediately upon entry, eliminating the need for plasmid transcription and translation that would otherwise be vulnerable to nuclease degradation
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 increases the production efficiency of genome-edited plants by achieving high frequencies of targeted mutations, reducing foreign DNA insertion, and enabling the regeneration of plants with stable genome edits, thus addressing regulatory concerns and improving agricultural applications.
Implementation Method 1
introducing a Cas protein and a guide RNA into an isolated plant protoplast to edit the genome of the plant protoplast
Implementation Method 2
as shown in human cells, RNA-guided engineered nuclease (RGEN) RNPs cleave chromosomal target sites immediately after transfection
Data Source
AI summary
The present invention relates to a method of increasing the production efficiency of gene-edited plants, regenerated from plant protoplasts, by use of a Cas protein-guide RNA ribonucleoprotein (RNP). According to the present invention, the method of increasing the production efficiency of gene-edited plants makes it possible to efficiently produce target gene-mutated plants and to minimize the introduction of foreign DNA into plants. Thus, the present invention can be very advantageously used in a wide variety of fields, including agriculture, food and biotechnology.