Complex Transgenic Trait Loci with Site-Specific DNA Integration
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Solution Overview
Problem
Existing methods for altering plant genomes lack the ability to precisely integrate multiple transgenic DNA sequences in defined regions, leading to unpredictable copy numbers and random integration, which hinders the production of fertile plants with desired genetic modifications.
Innovation Solution
A method involving the use of double-strand-break-inducing agents, such as endonucleases, to introduce targeted alterations at specific genomic locations, followed by identification and recovery of fertile plants with desired genetic alterations, and subsequent crossing to achieve plants with multiple linked modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If random transgene integration methods (Agrobacterium infection or biolistic particle bombardment) are used, then transformation efficiency is improved, but integration precision and copy number control deteriorate
Solution Approach 1:
The patent applies preliminary action by introducing double-strand breaks at specific genomic locations before transgene integration. This pre-established break pattern guides the transgene to integrate at predetermined sites rather than random locations, thereby achieving both high transformation efficiency and precise integration control simultaneously
Solution Approach 2:
The patent uses double-strand-break-inducing agents as intermediaries to mediate between the transgene and the genomic target site. These agents create controlled breaks that serve as integration landing zones, enabling precise positioning of transgenes while maintaining high transformation rates
2Manufacturing precision
If site-specific recombination is used to control transgene integration, then integration precision is improved, but the ability to produce fertile plants with multiple linked modifications deteriorates
Solution Approach 1:
The patent segments the genome modification process into multiple independent steps, each creating a specific alteration at a defined genomic location. By sequentially introducing double-strand breaks and integrating individual transgenes at separate target sites, the method produces fertile plants with multiple distinct genetic modifications that can be independently controlled and tracked
Solution Approach 2:
The patent employs dynamic control by adjusting the timing, sequence, and combination of double-strand-break-inducing agents applied to different target sites. This dynamic approach enables flexible production of plants with various combinations of genetic modifications while maintaining fertility and desired trait linkages
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
Enables the precise integration of multiple transgenic traits in defined genomic regions, resulting in fertile plants with desired genetic modifications, enhancing the control over transgene integration and promoting genetic linkage.
Implementation Method 1
The first double-strand-break-inducing agent is capable of inducing a first double-strand break in DNA comprising the first target sequence, and the second double-strand-break-inducing agent is capable of inducing a second double-strand break in DNA comprising the second target sequence
Implementation Method 2
It was shown that artificially induced site-specific genomic double-stranded breaks in plant cells were repaired by homologous recombination with exogenously supplied DNA
Data Source
AI summary
Methods for producing in a plant a complex transgenic trait locus comprising at least two altered target sequences in a genomic region of interest are disclosed. The methods involve the use of two or more double-strand-break-inducing agents, each of which can cause a double-strand break in a target sequence in the genomic region of interest which results in an alteration in the target sequence. Also disclosed are complex transgenic trait loci in plants. A complex transgenic trait locus comprises at least two altered target sequences that are genetically linked to a polynucleotide of interest. Plants, plant cells, plant parts, and seeds comprising one or more complex transgenic trait loci are also disclosed.