Maize Genome Targeted Insertion via Site-Specific Nucleases
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Solution Overview
Problem
Current methods for introducing a DNA of interest into a maize cell are inefficient and often result in random insertion, leading to variable expression and potential negative effects on agronomic performance.
Innovation Solution
The method involves identifying and targeting specific ideal genomic loci within the maize genome for precise integration of a DNA of interest using site-specific nucleases and nucleic acid molecules, ensuring high-expression and stable integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If random insertion methods are used to introduce DNA into maize cells, then the transformation process is simple, but the integration precision is low and expression stability is poor
Solution Approach 1:
The patent applies preliminary action by pre-identifying and characterizing ideal genomic target sites with specific features (unique sequences, appropriate chromatin structure, favorable epigenetic marks) before performing the transformation. This preliminary characterization of target loci enables precise targeting while maintaining manageable transformation procedures, resolving the contradiction between precision and complexity.
Solution Approach 2:
The patent uses site-specific nucleases (ZFNs, TALENs, CRISPR-Cas systems) as intermediary tools that mediate between the introduced DNA and the genomic target sites. These nucleases act as precise guides to direct integration to predetermined locations, achieving high integration precision without requiring overly complex transformation protocols.
2Manufacturing precision
If targeted insertion into predetermined genomic loci is performed, then integration precision is improved, but identifying suitable target sites becomes more difficult
Solution Approach 1:
The patent applies parameter changes by establishing specific criteria parameters for ideal target sites (sequence uniqueness, chromatin accessibility, epigenetic marks, distance from genes) and using these quantifiable parameters to identify suitable loci. This systematic parameter-based approach makes target site identification more straightforward and less difficult.
Solution Approach 2:
The patent performs preliminary identification and characterization of ideal target sites before the actual transformation process. By pre-mapping genomic loci that meet the established criteria and creating a catalog of suitable target sites, the difficulty of identifying appropriate targets during the transformation process is significantly reduced.
3Productivity
If non-ideal genomic sites are used for insertion, then transformation efficiency is high, but expression level and stability deteriorate
Solution Approach 1:
The patent applies local quality by identifying and selecting genomic loci with specific local characteristics that are favorable for both transformation and expression. Ideal target sites are characterized by unique sequences for precise targeting, appropriate chromatin structure for accessibility, and favorable epigenetic marks for stable expression. This localized optimization of genomic sites simultaneously achieves high transformation efficiency and reliable expression stability.
4Ease of manufacture
If random integration occurs, then the transformation process is straightforward, but agronomic performance may be negatively affected
Solution Approach 1:
The patent converts the potential harm of random integration into a benefit by using site-specific nucleases to direct integration to predetermined safe harbor loci. These target sites are specifically chosen to minimize disruption to endogenous genes while maintaining transformation efficiency. The approach transforms the randomness and potential harm into a controlled, beneficial outcome with improved agronomic performance.
Solution Approach 2:
The patent performs preliminary identification of ideal target sites that are unlikely to disrupt essential genes or regulatory elements. By pre-selecting safe harbor loci with favorable characteristics and avoiding regions with critical functions, the transformation process maintains simplicity while preventing harmful effects on agronomic performance.
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 the efficient and precise integration of a DNA of interest into the maize genome, resulting in high-quality transgenic events with minimal disruption to endogenous genes and improved agronomic performance.
Implementation Method 1
a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a genomic nuclease cleavage site
Data Source
AI summary
The present invention relates to methods and compositions for targeted insertion of polynucleotide molecules into ideal target sites in the genome of a maize plant. The present invention relates to maize recombinant molecules comprising heterologous sequences and also to methods of integrating a DNA of interest into a target maize genomic locus in a maize genome. The present invention also relates to regenerated maize plants or plant parts comprising the recombinant molecules and/or a DNA of interest.