Chromosomal Engineering via Oligonucleotide-Mediated End Joining
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Solution Overview
Problem
Current methods for chromosomal rearrangements in plant breeding are inefficient and lack control, leading to unintended genetic changes and reduced recombination frequencies, which hinder the introduction of desirable traits.
Innovation Solution
The use of CRISPR-Cas technology in combination with oligonucleotides that facilitate DNA end-joining, along with temperature treatments, to induce targeted chromosomal modifications such as inversions, deletions, and translocations in crop plant cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional chromosomal rearrangement methods are used, then chromosomal modifications can occur, but the efficiency is low and control is poor leading to unintended genetic changes
Solution Approach 1:
The patent applies preliminary action by introducing oligonucleotides with homology to chromosomal ends before the actual chromosomal rearrangement. These oligonucleotides pre-positioned DNA sequences that facilitate precise end-joining during the rearrangement process, thereby improving both efficiency and precision of chromosomal modification without causing unintended genetic changes
Solution Approach 2:
The patent uses oligonucleotides as intermediary molecules that mediate between the DNA break inducing agent and the chromosomal ends. These intermediaries bind to the chromosomal ends through homology and facilitate controlled DNA end-joining, enabling precise chromosomal rearrangements while preventing random mutations and improving overall precision
2Productivity
If large chromosomal rearrangements occur, then desirable traits can be introduced, but recombination frequencies are reduced and many genes are excluded from recombination
Solution Approach 1:
The patent applies local quality by making targeted chromosomal rearrangements at specific locations rather than large-scale random rearrangements. By using site-specific DNA break inducing agents and oligonucleotides with homology to specific chromosomal ends, the method achieves localized modifications that introduce desirable traits while preserving recombination frequencies in other regions of the chromosome
3Productivity
If DNA break inducing agents are used to create double strand breaks, then chromosomal modifications can be induced, but incorrect repair outcomes occur more frequently
Solution Approach 1:
The patent implements feedback by using oligonucleotides with homology to chromosomal ends that guide the repair process. The oligonucleotides provide sequence information that feedbacks to the repair machinery, ensuring correct alignment and joining of chromosomal ends, thereby reducing incorrect repair outcomes and improving reliability
Solution Approach 2:
The oligonucleotides serve as intermediary guides during DNA repair, mediating between the DNA break inducing agent and the chromosomal ends. They provide sequence specificity that ensures correct repair outcomes by facilitating homology-directed end-joining, thereby reducing incorrect repairs while maintaining high productivity
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 significantly enhances the efficiency and precision of chromosomal modifications, allowing for the controlled introduction of large genetic segments, thereby improving plant breeding outcomes.
Implementation Method 1
one or more oligonucleotides with substantial sequence similarity to one or more chromosomal ends at two different or distinct double strand break regions, and wherein the one or more oligonucleotides increase chromosomal modification efficiency at the genomic target site by facilitating DNA end-joining
Implementation Method 2
the plant cell is incubated at a temperature of at least about 28° C. such that the higher temperature effect increases chromosomal modification efficiency at the genomic target site by facilitating DNA end-joining
Implementation Method 3
Newer technologies utilizing archaeal or bacterial adaptive immunity systems have been identified, called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which comprise different domains of effector proteins that encompass a variety of activities (DNA recognition, binding, and optionally cleavage)
Data Source
AI summary
Compositions and methods are provided for high-efficiency large scale manipulation of genomic regions and chromosomal engineering of plant genomes. Enhancement of chromosomal modification includes the provision of one or more linker oligonucleotides complementary to the distinct chromosomal ends following double strand breaks, increasing the temperature effect and/or recurrent cutting by an endonuclease to facilitate higher chromosomal modification including large segment translocations and recombination. Site-specific directed DNA breaks under one or more of the experimental conditions (including CRISPR-Cas systems) disclosed enhance targeted recombination frequencies, crossover efficiency and movement of large chromosomal segments in crop plant cells.