CRISPR-Associated Transposases for Precise Plant Genome Integration
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Solution Overview
Problem
Current genome editing techniques in plants face challenges such as undesirable byproducts from double-strand breaks and inefficient homology-directed repair, limiting the ability to introduce desired genetic traits effectively.
Innovation Solution
The use of an RNA-guided DNA binding complex and a transposition complex, combined with a donor polynucleotide flanked by transposon end sequences, to integrate a cargo sequence into plant genomes, leveraging autonomous replicons from geminiviruses for enhanced efficiency and precision.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If double-strand breaks are used for genome editing, then DNA insertion can be achieved, but undesirable byproducts including indels, large-scale deletions, and chromosomal translocations are generated
Solution Approach 1:
The patent converts the harmful effect of double-strand breaks into a beneficial process by using the breaks to facilitate precise DNA insertion through HDR. The DSBs are not merely tolerated but are strategically induced to create insertion sites, while the harmful byproducts are minimized through optimized repair templates and delivery methods. This transforms the previously harmful DSB mechanism into a controlled tool for precise genetic modification.
Solution Approach 2:
The patent introduces homology-directed repair (HDR) as an intermediary mechanism between the double-strand break and the final DNA insertion. The HDR process acts as a mediator that guides the repair of DSBs in a controlled manner, using provided repair templates to achieve precise insertions rather than random mutations. This intermediary mechanism filters out the harmful byproducts while maintaining the beneficial insertion outcome.
2Manufacturing precision
If homology-directed repair is used for DNA insertion, then targeted integration can be achieved, but the process is limited to S and G2 phases and requires sufficient repair template supply
Solution Approach 1:
The patent applies preliminary action by pre-providing repair templates and preparing the genomic site before the actual insertion event. The repair templates are designed in advance with appropriate homology arms and cargo sequences, and the delivery system is optimized to ensure template availability in the correct cell cycle phase. This preliminary preparation enables HDR to proceed efficiently even with the cell cycle limitations, as the necessary components are already in place when the cell enters the permissive S/G2 phases.
3Productivity
If conventional genome editing tools are used, then genetic traits can be introduced, but the efficiency and reliability are insufficient for effective trait integration
Solution Approach 1:
The patent merges multiple conventional tools and mechanisms into a unified system that combines CRISPR/Cas9 for targeted DSB creation, HDR for precise repair and insertion, and optimized delivery vectors for efficient gene transfer. This integration of multiple functions into a single coordinated process significantly improves both the efficiency and reliability of trait integration, as the components work synergistically rather than independently. The merged system eliminates the need for separate steps and reduces the overall failure rate associated with conventional separate approaches.
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 and efficient integration of genetic cargo sequences, reducing undesirable mutations and improving traits like herbicide tolerance, drought resistance, and modified metabolic pathways in plants.
Implementation Method 1
an RNA-guided DNA binding complex or one or more first helper polynucleotides each comprising a sequence encoding a component of the RNA-guided DNA binding complex, wherein the RNA-guided DNA binding complex comprises one or more Cas proteins, a transposase, a crRNA
Implementation Method 2
a transposition complex or one or more second helper polynucleotides each comprising a sequence encoding a component of the transposition complex, wherein the transposition complex comprises one or more transposases and one or more transposons
Data Source
AI summary
Disclosed herein include methods, compositions, and kits suitable for use in integration of a nucleic acid sequence into a double-stranded target sequence of a genome of a plant cell or a target plasmid in a plant cell. In some embodiments, the methods, systems and nucleic acid compositions comprise polynucleotides encoding and RNA-guided DNA binding complex, a transposition complex, for insertion of cargo sequence using transposition.