HDR Efficiency via Donor Localization and Cell Cycle Regulation
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Solution Overview
Problem
Current methods for targeted genome editing face challenges in controlling whether a double-stranded break (DSB) is repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ), with HDR being less efficient than NHEJ.
Innovation Solution
The development of methods that involve contacting cells with or expressing specific components, such as engineered DNA binding domains (DBDs) and nucleases, to enhance the efficiency of HDR by localizing donor molecules to the site of DSBs, regulating nuclease activity by cell cycle phases, and recruiting or blocking DNA repair factors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If HDR is used to repair DSBs, then precise genome editing is achieved, but the repair efficiency is lower compared to NHEJ
Solution Approach 1:
The patent applies preliminary action by expressing HDR-enhancing factors (such as Rad51, BRCA1, or other homologous recombination proteins) before or at the time of DSB induction. This pre-preparation of the cellular machinery ensures that when the DSB occurs, the HDR pathway is already primed and ready to act, thereby increasing HDR efficiency without compromising precision. The preliminary expression of these factors shifts the repair pathway preference from NHEJ to HDR before the repair event occurs.
Solution Approach 2:
The patent employs parameter changes by modifying cellular conditions to favor HDR over NHEJ. This includes changing the expression levels of key repair proteins, adjusting the cell cycle phase (since HDR is active in S/G2 phases), or introducing exogenous factors that enhance HDR machinery activity. By altering these parameters, the cellular repair pathway preference is shifted from the default NHEJ to HDR, improving both precision and efficiency of genome editing.
2Productivity
If NHEJ is used to repair DSBs, then repair efficiency is high, but precise sequence changes cannot be achieved
Solution Approach 1:
The patent applies preliminary anti-action by using inhibitors or interfering RNAs to block NHEJ pathway components (such as Ku70/Ku80, DNA-PK, or Ligase IV) before DSB repair occurs. By preemptively inhibiting the high-efficiency but imprecise NHEJ pathway, the cell is forced to use the slower but precise HDR pathway. This counter-action against the dominant repair mechanism enables precise sequence changes to be achieved while maintaining reasonable repair efficiency.
3Manufacturing precision
If HDR efficiency is increased, then precise genome editing improves, but the complexity of the method increases
Solution Approach 1:
The patent applies universality by using a platform approach where a single HDR-enhancing factor or a small set of factors can be used across multiple different genome editing targets and contexts. For example, overexpression of a universal HDR protein like Rad51 or BRCA1 can enhance HDR efficiency at multiple different DSB sites throughout the genome, eliminating the need for target-specific optimization. This multi-functional approach simplifies the overall method while maintaining high precision across diverse applications.
Data Source
AI summary
The present invention relates to methods to improve the absolute rate of homology-directed repair (HDR) and/or to improve the relative rate of HDR compared with non-homologous end joining (NHEJ).