Genome Editing Compounds Enhancing HDR via Cell Cycle Synchronization
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Solution Overview
Problem
Current approaches for precise genome editing, especially in stem cells, have low efficiencies ranging from 0.5-15% due to the error-prone nature of Non-homologous-End-Joining (NHEJ) and the challenges in promoting Homology Directed Repair (HDR).
Innovation Solution
The use of specific combinations of compounds, including inhibitors of histone deacetylase (HDAC), NEDD8 activating enzyme (NAE), DNA-dependent Protein Kinase (DNA-PK), and Replication Protein A (RPA), along with a catalytically inactive but structurally intact DNA-PKcs subunit, to enhance the efficiency of precise genome editing by promoting HDR and suppressing NHEJ.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR Cas9 is used to introduce double-stranded breaks for genome editing, then gene knockout efficiency is improved, but off-target mutations increase and precise editing efficiency remains low
Solution Approach 1:
The invention divides the single Cas9 nuclease into two separate nickase components (Cas9n1 and Cas9n2), each capable of introducing single-stranded nicks. By using two separate guide RNAs (gRNA1 and gRNA2) that target opposite strands, the system achieves double-stranded break only at the intended target site where both nicks occur in close proximity, while minimizing off-target effects from individual nicks
Solution Approach 2:
The invention introduces a donor DNA template as an intermediary element that facilitates precise repair through homology-directed repair (HDR). The donor template contains the desired genetic modification flanked by homology arms that guide precise integration at the target site, replacing error-prone NHEJ with accurate HDR-mediated editing
2Speed
If Non-homologous-End-Joining (NHEJ) pathway is used for DNA repair, then rapid repair is achieved, but editing precision deteriorates due to error-prone insertions and deletions
Solution Approach 1:
The invention changes the repair pathway parameters by providing a donor DNA template with homology arms, which shifts the repair mechanism from NHEJ to HDR. This parameter change (presence of homology template) transforms the repair outcome from error-prone indels to precise editing, while the nickase approach maintains the speed advantage by creating controlled single-strand breaks rather than requiring complex double-strand break formation
3Manufacturing precision
If Homology Directed Repair (HDR) is promoted for precise editing, then editing precision is improved, but repair efficiency decreases compared to NHEJ
Solution Approach 1:
The invention performs preliminary action by pre-synchronizing cells to the S/G2 phase of the cell cycle before introducing the editing components. This preliminary timing ensures that HDR machinery is naturally upregulated and available, significantly enhancing HDR efficiency without compromising precision. The cell cycle synchronization prepares the cellular environment in advance to favor HDR over NHEJ
4Manufacturing precision
If cell cycle synchronization to S/G2 phase is performed to enhance HDR, then precise editing efficiency is improved, but process complexity and time increase
Solution Approach 1:
The invention employs periodic action through cell cycle synchronization, cycling cells through specific phases (S/G2) at predetermined intervals before editing. This periodic timing strategy maximizes HDR efficiency by ensuring cells are in the optimal phase for homology-directed repair, while the rhythmic nature of cell cycle control makes the process manageable and reproducible despite the added temporal complexity
Data Source
AI summary
The present invention relates to compounds, compositions and kits suitable to precise genome editing efficiency in a eukaryotic target organism.