Site-Specific Integration in Plant Genomes via Double-Strand Breaks
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Solution Overview
Problem
Current plant transformation methods, such as Agrobacterium infection and biolistic particle bombardment, result in random and unpredictable integration of transgenes into the plant genome, limiting the ability to target specific genomic positions and stack additional polynucleotides effectively.
Innovation Solution
The development of methods and compositions for introducing transgenic target sites for Site-Specific Integration (SSI) into specific double-strand-break inducing agent target sites within a genomic window of a plant genome, using agents like Cas9 endonuclease, to achieve targeted integration of polynucleotides of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If random integration methods (Agrobacterium infection, biolistic particle bombardment) are used, then the transformation process is simple and widely applicable, but the integration position of transgenes is unpredictable and cannot be targeted to specific genomic positions
Solution Approach 1:
The patent divides the transformation system into separate functional components: a donor DNA molecule containing the transgene flanked by homology arms, and a targeting system using site-specific recombination or homology-directed recombination. This segmentation allows independent optimization of each component while achieving precise integration.
Solution Approach 2:
The patent employs intermediary mechanisms such as site-specific recombination systems (e.g., Cre-lox, FRT) or homology-directed recombination as mediators between the donor DNA and the target genomic site. These intermediaries facilitate controlled integration at predetermined locations, resolving the contradiction between precision and complexity.
2Adaptability or versatility
If random integration methods are used, then the transformation process is straightforward, but the ability to stack additional polynucleotides of interest near desired integration sites is limited
Solution Approach 1:
The patent implements a nested structure where multiple polynucleotides of interest are arranged within a single donor DNA molecule, with each polynucleotide flanked by homology arms targeting specific genomic positions. This nesting allows simultaneous integration of multiple elements at predetermined locations, enabling stacking while maintaining precision.
Solution Approach 2:
The patent performs preliminary design and arrangement of multiple polynucleotides and their corresponding homology arms in the donor DNA before transformation. This preliminary planning ensures that all polynucleotides will integrate at their predetermined positions simultaneously, enabling stacking capability without sacrificing position control.
3Productivity
If random integration methods are used, then the transformation process is simple, but producing a fertile plant with altered genome is difficult and inefficient
Solution Approach 1:
The patent applies local quality by introducing homology arms with specific sequence characteristics matched to the target genomic location. This local customization ensures reliable and reproducible integration at the desired site, thereby improving the reliability of genome alteration and subsequent fertile plant production.
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 precise targeting of transgenic sequences into desirable genomic positions, enabling the creation of complex trait loci and ensuring the integration of multiple transgenic elements independently, thereby improving the efficiency and precision of plant genome modification.
Implementation Method 1
introducing transgenic target sites for Site Specific Integration (SSI) into at least one target site of a double-strand-break inducing agent
Data Source
AI summary
Compositions and methods are provided for introducing transgenic target sites for Site Specific Integration (SSI) and/or polynucleotides of interest into at least one double-strand break target site of a double-strand-break inducing agent in a genomic window of a plant genome. Also provided are methods and compositions for producing a complex trait locus in a genomic window of a plant comprising at least one transgenic target site for site specific integration integrated in at least double-strand-break target site. The double-strand-break target site can be, but is not limited to, a target site for a zinc finger endonuclease, an engineered endonuclease, a meganuclease, a TALENs and/or a Cas endonuclease. The genomic window of said plant can comprise at least one genomic locus of interest such as a trait cassette, a transgene, a mutated gene, a native gene, an edited gene or a site-specific integration (SSI) target site.