Multipartite CRISPR Donor Assembly for Multiplex Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas9 and CRISPR-Cpf1 systems require multiple specific donor polynucleotides for genome editing, making multiplex approaches laborious and inefficient, especially for screening multiple targets or modifications in eukaryotic cells.
Innovation Solution
A method involving the use of at least two double-stranded polynucleotides that can assemble into a double-stranded polynucleotide construct with sequence identity to the genome, integrating into the genome near a break site, allowing for targeted genome editing with reduced need for multiple specific donor polynucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If multiple specific donor polynucleotides are used for each target site in CRISPR genome editing, then precise targeted modification can be achieved, but the experimental work becomes laborious and complex especially for multiplex approaches
Solution Approach 1:
The patent applies universality by designing a single donor polynucleotide template that can serve multiple target sites simultaneously. The donor template contains multiple candidate insertion sequences that can be integrated at different genomic locations through CRISPR-Cas9 mediated homology-directed repair, eliminating the need to design and synthesize separate donor polynucleotides for each target site.
Solution Approach 2:
The patent segments the donor polynucleotide template into distinct functional regions: a constant backbone sequence and multiple candidate insertion sequences. This segmentation allows the single template to provide precise targeted modification at multiple locations while reducing experimental complexity by using one synthesized polynucleotide instead of multiple site-specific donors.
2Adaptability or versatility
If multiple donor polynucleotides are synthesized and transformed for screening multiple targets, then comprehensive genome editing can be achieved, but the time and resources required increase significantly
Solution Approach 1:
The patent applies preliminary action by pre-designing the donor polynucleotide template with multiple candidate insertion sequences that can address multiple target sites. This preliminary preparation of a multi-functional donor template eliminates the need for sequential synthesis and transformation of multiple site-specific donors, significantly reducing experimental time while maintaining multiplex screening capability.
Solution Approach 2:
The patent merges multiple donor polynucleotide functions into a single integrated template. By combining multiple candidate insertion sequences within one donor polynucleotide structure, the system enables simultaneous or sequential editing of multiple target sites without requiring separate transformation events for each donor, thus reducing both time and resource requirements.
3Device complexity
If a single donor polynucleotide template with multiple candidate insertion sequences is used, then experimental work is simplified, but the ability to achieve precise targeted modification at each specific site may be compromised
Solution Approach 1:
The patent applies local quality by ensuring that each candidate insertion sequence within the donor polynucleotide template contains the specific homology arms and genetic information required for precise integration at its corresponding target site. Each local region of the donor template is optimized for its specific target, maintaining site-specific editing precision even though the overall structure is unified and simplified.
Data Source
AI summary
The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-assembly gene editing system in a eukaryotic cell.