Donor Nucleic Acid Design for Higher Homologous Recombination
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Solution Overview
Problem
Existing genome-editing systems face low efficiency in homologous recombination, requiring laborious and time-consuming screening of clonal cells to identify those with the desired genotype.
Innovation Solution
A method involving a CRISPR complex to create double-strand breaks in cells, followed by targeted delivery of donor nucleic acid molecules with matched termini and nuclease-resistant groups, optimizing the timing and amount of donor nucleic acid introduction to enhance homologous recombination efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional genome-editing systems are used to perform homologous recombination, then genetic modification can be achieved, but the efficiency is low requiring extensive screening of clonal cells
Solution Approach 1:
The patent modifies key parameters of the homologous recombination process including: (1) optimizing the ratio of donor nucleic acid to target locus, (2) adjusting the timing of donor nucleic acid delivery relative to CRISPR complex formation, (3) modifying donor nucleic acid structure with asymmetric termini and nuclease-resistant groups, and (4) controlling the concentration of CRISPR components. These parameter changes collectively enhance homologous recombination efficiency to at least 20% of cells, dramatically reducing the time and effort required to identify correctly edited cells.
2Ease of operation
If donor nucleic acid is delivered simultaneously with CRISPR complex, then the process is simplified, but homologous recombination efficiency decreases
Solution Approach 1:
The patent implements a two-stage delivery process where the CRISPR complex is introduced into cells first to establish the double-strand break and prepare the target locus. After a controlled interval (optimally 1-4 hours), the donor nucleic acid is then delivered. This preliminary action of creating the break site before introducing the repair template maximizes homologous recombination efficiency, achieving at least 20% edited cells while maintaining operational simplicity through standardized timing protocols.
3Device complexity
If standard donor nucleic acid molecules are used, then the system is simpler, but homologous recombination efficiency is limited
Solution Approach 1:
The patent applies local quality modifications to specific regions of the donor nucleic acid molecule: (1) asymmetric termini design where the 5' and 3' ends have different structures and lengths to match the polarity of the double-strand break, (2) incorporation of nuclease-resistant chemical groups (such as phosphorothioate modifications) at critical positions to protect against degradation, and (3) optimization of homology arm lengths and sequences at the termini. These localized structural enhancements significantly improve homologous recombination efficiency to at least 20% of cells while maintaining overall system simplicity.
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
Enhances homologous recombination efficiency to at least 20% of cells, reducing the time and effort required to identify correctly edited cells.
Implementation Method 1
contacting the population of cells with a nucleic acid cutting entity under conditions that allow for the generation of double-stranded break at a target locus in nucleic acid present inside cells
Implementation Method 2
contacting the cells with the donor nucleic acid molecule under conditions that allow for homologous recombination to occur at the target locus in at least 20% of the cells
Data Source
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AI summary
The present disclosure generally relates to compositions and methods for improving the efficiency of homologous recombination. In particular, the disclosure relates to reagents and the use of such reagents.