Mobile RNA Vector for Meristem Genome Editing
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Solution Overview
Problem
Current methods for achieving targeted genetic modifications in plant meristems are inefficient and often require transgenic plants, which are difficult to produce and may not be suitable for commercialization, especially for crops like bell pepper where meristem formation is hard to trigger in vitro.
Innovation Solution
A vector expressing a coding RNA with a CRISPR-nuclease and a mobile element, such as a virus or naked DNA, is used to facilitate intercellular translocation to meristem cells, enabling targeted genomic modifications without the need for regeneration or transgenic plants, using elements like Tobacco Rattle Virus (TRV) or Tobacco Mosaic Virus (TMV) for delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If direct delivery of transgenes to meristem is attempted, then heritable mutations can be achieved, but the method is difficult and only successful in limited plant species
Solution Approach 1:
The patent uses mobile genetic elements (transposons, retrotransposons, or viral vectors) as intermediaries to deliver CRISPR-Cas9 components to meristem cells. These mobile elements naturally move between cells and can deliver therapeutic genes without requiring direct transgene delivery methods, thus resolving the contradiction between achieving heritable mutations and the difficulty of delivery.
Solution Approach 2:
The patent employs the plant's own mobile genetic elements and natural translocation mechanisms to deliver the editing components to meristem cells. By harnessing the plant's intrinsic biological systems rather than imposing external delivery methods, the approach achieves both reliability and ease of manufacture across diverse plant species.
2Reliability
If Cas9 transgenic plants are used for heritable mutations, then gene editing can be achieved, but the plants express transgenic Cas9 protein which complicates commercialization
Solution Approach 1:
The patent extracts only the necessary CRISPR guide RNA component while leaving out the Cas9 protein-encoding transgene. By using mobile genetic elements to deliver only the guide RNA or a transient CRISPR system that degrades after editing, the method achieves heritable mutations without leaving persistent transgenic Cas9 protein in the plant, thus resolving the contradiction between mutation heritability and commercialization safety.
Solution Approach 2:
The patent performs the CRISPR editing action preliminarily through mobile genetic elements that deliver editing components temporarily. The editing occurs before the plant completes its life cycle, and the transgenic elements are designed to be transient or self-limiting, ensuring mutations are heritable while minimizing residual transgenic protein expression.
3Adaptability or versatility
If in vitro tissue culture is used to trigger meristem formation, then edited somatic cells can be converted to edited meristems, but this method does not work in crops where meristem formation is hard to trigger
Solution Approach 1:
The patent uses mobile genetic elements as intermediaries that can deliver CRISPR components directly to meristem cells in vivo, bypassing the need for in vitro tissue culture and artificial meristem induction. This approach works across diverse crop species regardless of their responsiveness to tissue culture protocols, thus resolving the contradiction between versatility and ease of manufacture.
Solution Approach 2:
Instead of trying to induce meristem formation from edited somatic cells through difficult in vitro culture, the patent inverts the approach by directly delivering editing components to naturally occurring meristem cells in the plant. This reversal eliminates the need for challenging tissue culture protocols while maintaining the ability to generate heritable mutations.
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 allows for efficient and heritable genetic modifications in plant meristems, bypassing the need for regeneration and transgenic plants, ensuring that edits are transmitted to subsequent generations while avoiding the use of transgenic Cas9 proteins, thus making it feasible for commercial crops.
Implementation Method 1
the mobile element enables intercellular translocation of the coding RNA
Implementation Method 2
facilitate intercellular translocation to meristem cells, enabling targeted genomic modifications
Data Source
AI summary
The invention concerns the targeted genomic modification of a plant cell, preferably a meristem cell. More in particular, the invention pertains to a vector expressing a coding RNA, wherein the coding RNA comprises a sequence encoding a CRISPR-nuclease and a mobile element, wherein the mobile element enables intercellular translocation of the coding RNA, preferably intercellular translocation to a meristem cell. The invention further concerns an editing RNA comprising the coding RNA and further comprising a guide RNA.