Modified Agrobacterium for Precise Plant Genome Editing
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Solution Overview
Problem
Current methods for plant genetic modification, such as those using Agrobacterium, often result in the integration of exogenous DNA into the plant genome, leading to transgenic plants. This raises concerns among consumers and regulators, and there is a need for more precise and efficient methods to introduce desired mutations in plant cells without integrating foreign DNA.
Innovation Solution
A modified bacterium with reduced VirD5 activity and carrying site-specific DNA editing machinery is used to introduce precise mutations into plant cells. This bacterium employs transient expression of DNA-editing agents from the T-DNA, minimizing T-DNA integration and allowing for the generation of gene-edited, non-transgenic plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If Agrobacterium is used to transfer DNA to plant cells for genetic engineering, then the ability to introduce desired mutations is improved, but integration of exogenous DNA into the plant genome occurs leading to transgenic plants which raises consumer and regulatory concerns
Solution Approach 1:
The invention extracts and removes the harmful component (exogenous DNA integration) while preserving the useful function (mutation introduction). By using modified Agrobacterium with reduced VirD5 activity, the bacteria transfer only the minimal necessary genetic material for editing without integrating large portions of exogenous DNA, thus taking out the harmful integration effect while maintaining the beneficial editing capability
Solution Approach 2:
The invention changes the parameter of VirD5 activity from normal levels to reduced levels. This parameter change in the Agrobacterium's virulence machinery alters the transfer behavior, resulting in reduced T-DNA integration while still enabling sufficient transfer of the editing machinery components needed for precise mutation introduction
2Object-generated harmful factors
If VirD5 activity is reduced in the bacterium, then T-DNA integration into the plant genome is minimized, but the efficiency of DNA transfer and editing machinery delivery must be maintained
Solution Approach 1:
The invention applies partial action by reducing VirD5 activity to a level that is sufficient for delivering the editing machinery components but insufficient for causing extensive T-DNA integration. This partial reduction achieves the desired balance where enough genetic material is transferred to enable editing, but not enough to cause harmful integration events
3Ease of manufacture
If exogenous DNA sequence is cloned into T-DNA for integration into plant genome, then the plant expresses new protein from integrated DNA, but this creates transgenic plants with integrated foreign DNA
Solution Approach 1:
The invention uses the Agrobacterium as an intermediary that delivers editing machinery (Cas9, guide RNA, and a minimal DNA sequence) rather than delivering the entire exogenous DNA sequence for integration. The bacterium mediates the transfer of only the essential components needed for in planta editing, avoiding the need for large-scale T-DNA integration while still achieving the goal of introducing new genetic functions
Data Source
AI summary
The present invention relates to modified bacteria for introducing desired mutations in target sequences in plant cells, wherein the bacteria has reduced VirD5 activity and carries site-specific DNA editing machinery. The invention also provides methods for generating plant cells, plant parts, plants or populations thereof using such bacteria.