Monocot Genome Editing via Double-Strand Break Induction
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Solution Overview
Problem
Current methods for site-specific modification of plant genomes often fail to produce fertile plants with desired alterations, as they lack efficient control over transgene integration and repair mechanisms.
Innovation Solution
A method involving contacting monocot plant cells with a phenotypic marker, a DNA fragment, and a double-strand break inducing agent to create targeted alterations at specific recognition sequences, allowing for selection and identification of cells with specific genome modifications without using a phenotypic marker, and recovering fertile plants with these alterations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If homologous recombination-based transgene integration is used, then site-specific modification is achieved, but the frequency of recovering fertile plants with desired alterations is low
Solution Approach 1:
The patent applies preliminary action by inducing double-strand breaks at target genomic locations before introducing the DNA fragment. This pre-prepared break site facilitates subsequent homologous recombination, thereby increasing the efficiency of site-specific modification and the frequency of recovering fertile plants with desired alterations
2Ease of manufacture
If random transgene integration is used, then transformation is simpler, but integration occurs at unpredictable locations with uncontrollable copy number
Solution Approach 1:
The patent uses homologous recombination as an intermediary mechanism between the introduced DNA fragment and the genomic target site. By requiring sequence homology between the fragment and target, this intermediary process ensures precise integration at predetermined locations while maintaining relative transformation simplicity
3Manufacturing precision
If site-specific recombination is used, then transgene integration location is controlled, but the method complexity increases
Solution Approach 1:
The patent employs the cell's own homologous recombination repair machinery to achieve site-specific integration. By leveraging the organism's endogenous repair systems rather than introducing complex external recombination enzymes, the method achieves precise integration control while minimizing additional procedural complexity
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 increases the frequency of recovering fertile monocot plants with desired genome alterations, such as insertions, deletions, or replacements, compared to control methods, using techniques like PCR, Southern blot, and DNA sequencing for identification.
Implementation Method 1
a double-strand break inducing agent capable of inducing a double-strand break at the recognition sequence
Implementation Method 2
selecting cells comprising the phenotypic marker
Implementation Method 3
using techniques like PCR, Southern blot, and DNA sequencing for identification
Implementation Method 4
using techniques like PCR, Southern blot, and DNA sequencing for identification
Implementation Method 5
using techniques like PCR, Southern blot, and DNA sequencing for identification
Data Source
AI summary
Methods and compositions for altering the genome of a monocot plant cell, and a monocot plant are disclosed. The methods and compositions use a double-strand break inducing agent to alter a monocot plant or plant cell genomic sequence comprising a recognition sequence for the double-strand break inducing agent.