TRV Viral Vector for CRISPR Plant Genome Editing
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Solution Overview
Problem
Current methods for targeted genetic modification in plants face inefficiencies due to the homology-directed repair process and lack of efficient delivery methods for genome-engineering reagents, particularly for stable expression of CRISPR/Cas9 systems, which hinders the discovery and development of novel agricultural traits.
Innovation Solution
A viral-mediated genome-editing platform using the Tobacco Rattle Virus (TRV) to deliver guide RNA molecules and the Cas9 endonuclease into plant cells, facilitating targeted genome editing and multiplexing without the need for stable transformation, enabling systemic delivery and germinal transmission of genomic modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If stable transformation methods are used to deliver CRISPR/Cas9 reagents into plant cells, then the expression of Cas9 and gRNA molecules can be maintained, but the process is time-consuming and labor-intensive requiring generation of transformant lines and monitoring in subsequent generations
Solution Approach 1:
The patent uses Agrobacterium tumefaciens as an intermediary vector to deliver CRISPR/Cas9 reagents into plant cells. The bacteria serve as a temporary delivery vehicle that transfers the genetic material without requiring stable transformation of the plant genome, thus avoiding the time-consuming process of generating transformant lines while still achieving reliable expression of Cas9 and gRNA molecules in the plant cells
Solution Approach 2:
The patent performs preliminary delivery of CRISPR/Cas9 reagents through Agrobacterium-mediated transformation before the actual genome editing process. By pre-delivering the necessary components (Cas9 gene, gRNA) into the plant cells using the bacterial vector, the system prepares the editing machinery in advance, allowing subsequent generations to inherit the modifications without requiring repeated stable transformation processes
2Manufacturing precision
If protein engineering is used to customize genome-editing platforms like ZFNs and TALENs, then sequence specificity can be achieved, but the process is time-consuming and labor-intensive
Solution Approach 1:
The patent uses CRISPR RNA (crRNA) as a copyable genetic template that directly encodes the sequence-specific targeting information. Instead of engineering proteins for each target sequence, the system copies the desired specificity through RNA transcription from DNA templates, allowing rapid customization by simply changing the nucleotide sequence of the crRNA guide region rather than performing complex protein engineering
Solution Approach 2:
The patent replaces the mechanical/protein-based targeting system (where amino acid sequences in ZFNs and TALENs physically interact with DNA) with a nucleic acid-based system. The crRNA guide sequence hybridizes to the target DNA through base-pairing rules, substituting the complex protein-DNA recognition mechanism with a simpler RNA-DNA hybridization process that is easier to manufacture and customize
3Quantity of substance
If current delivery methods are used for genome-engineering reagents, then the reagents can be introduced into plant cells, but the efficiency of delivery is insufficient for effective trait discovery and development
Solution Approach 1:
The patent employs Agrobacterium tumefaciens as a natural delivery intermediary that has evolved mechanisms to efficiently transfer DNA into plant cells. This biological mediator utilizes the bacteria's natural transformation capabilities to introduce CRISPR/Cas9 reagents at high efficiency, overcoming the limitations of artificial delivery methods and enabling effective trait discovery and development through improved reagent delivery
Data Source
AI summary
The present disclosure provides a viral-mediated genome-editing platform that facilitates multiplexing, obviates stable transformation, and is applicable across plant species. The RNA2 genome of the tobacco rattle virus (TRV) was engineered to carry and systemically deliver a guide RNA molecules into plants overexpressing Cas9 endonuclease. High genomic modification frequencies were observed in inoculated as well as systemic leaves including the plant growing points. This system facilitates multiplexing and can lead to germinal transmission of the genomic modifications in the progeny, thereby obviating the requirements of repeated transformations and tissue culture. The editing platform of the disclosure is useful in plant genome engineering and applicable across plant species amenable to viral infections for agricultural biotechnology applications.