Concatemer Donor Design for CRISPR HDR Efficiency
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Solution Overview
Problem
Current genome-editing techniques, such as CRISPR/Cas, face limitations in the frequency and efficiency of homology-directed repair (HDR) in certain organisms, particularly in plants, which hampers targeted genome editing and genetic pathway engineering.
Innovation Solution
The use of a concatemer design where multiple copies of donor or template DNA sequences, flanked by guide RNA targets, are introduced into cells to increase the availability of repair templates and enhance the frequency of HDR by providing multiple copies of heterologous polynucleotides for integration into double-strand breaks, facilitated by Cas endonucleases and guide RNA molecules.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple copies of donor DNA sequences flanked by guide RNA targets are introduced into cells, then the frequency of homology-directed repair increases, but the device complexity increases
Solution Approach 1:
The donor DNA is divided into multiple copies, each flanked by guide RNA target sequences. The Cas9-guide RNA complex segments the donor DNA by cleaving at these target sites, releasing individual repair templates that can then be integrated into the double-strand break. This segmentation increases the availability of repair templates and enhances HDR frequency while managing complexity through modular design.
Solution Approach 2:
The guide RNA target sequences are pre-flanked onto the donor DNA sequences before introduction into the cell. This preliminary arrangement ensures that when the Cas9-guide RNA complex is introduced, it can immediately cleave the donor DNA at the correct sites to release repair templates, eliminating the need for additional processing steps and streamlining the HDR process.
2Quantity of substance
If multiple copies of heterologous polynucleotides are provided for integration, then the availability of repair templates increases, but the manufacturing precision requirements increase
Solution Approach 1:
Multiple identical or variant copies of the donor DNA sequence are introduced, each flanked by guide RNA target sequences. These copies serve as templates for homology-directed repair. The copying approach increases the quantity of available repair templates, improving the probability of successful HDR, while the standardized structure of each copy maintains manufacturing precision through consistent design elements.
Solution Approach 2:
The invention allows for variations in the donor DNA sequences between copies (e.g., different heterologous polynucleotides, different flanking sequence lengths or compositions). By changing parameters such as sequence content, length, or composition while maintaining the overall structure, the system increases template diversity and availability without compromising the precision of the HDR process at the target site.
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 increases the frequency of homology-directed repair, enabling more efficient targeted genome editing and genetic modifications in plants by increasing the in vivo load of repair templates and improving the accessibility of these templates to the DNA target site.
Implementation Method 1
a guide RNA molecule that forms a complex with the Cas endonuclease to create a double-strand break in the target polynucleotide
Implementation Method 2
cleaving the plurality of sequence units with the complex to release the heterologous polynucleotides
Implementation Method 3
Repair of a double-strand break can be via Non-Homologous End Joining (NHEJ) or Homology-Directed Repair (HDR)/Homologous Recombination (HR)
Data Source
AI summary
Methods and compositions are provided for the improvement of homology-directed repair of a double strand break in a plant cell, using concatemers of heterologous polynucleotides that are flanked by sequences capable of sequence hybridization with a guide RNA. In some aspects, the double strand break is created by an RNA-guided Cas endonuclease. The homology-directed repair of the double-strand break may include incorporation of a heterologous polynucleotide, for example a gene encoding a trait of agronomic importance. The homology-directed repair of the double-strand break may occur as a result of template-directed repair using a heterologous polynucleotide as a repair template.