Knock-In Cell Production Using Combined NHEJ and Homologous Repair
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Solution Overview
Problem
Existing genome editing methods struggle to insert long strands of donor DNA into a genome with high efficiency and accuracy, particularly in mammalian cells and fertilized eggs, due to the dominance of non-homologous end joining and rarity of homologous recombination.
Innovation Solution
A site-specific nuclease system is used to combine non-homologous end joining and homologous recombination by targeting and cleaving specific sequences in the donor DNA and genomic DNA, allowing for the integration of long donor sequences through combinational repair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If a large number of cells are cultured to obtain sufficient cells for analysis, then the quantity of cells increases, but the time required for cell culture and the risk of bacterial contamination increase
Solution Approach 1:
The patent applies preliminary action by pre-integrating the selectable marker gene and loxP sites into the target gene locus before cell culture. This allows for rapid enrichment and identification of knock-in cells without requiring long-term culture of large cell populations, thereby reducing both time and contamination risk while still obtaining sufficient cells for analysis
2Reliability
If conventional methods are used to produce knock-in cells, then the process follows standard procedures, but the time required is excessive and bacterial contamination occurs
Solution Approach 1:
The patent implements feedback mechanisms through selectable markers that enable rapid identification and enrichment of successfully modified cells. The puromycin resistance marker provides immediate feedback on transfection success, and the FRT-flanked marker system allows for subsequent selection against random integrants, significantly reducing production time while maintaining reliability
Solution Approach 2:
The patent uses intermediary elements including the FRT site and Flp recombinase system as mediators to achieve precise knock-in. These intermediaries facilitate the removal of selectable markers after initial selection, enabling rapid enrichment of true knock-in cells without requiring prolonged culture periods, thus reducing time while maintaining process reliability
3Quantity of substance
If the number of cultured cells is increased to ensure sufficient cells for analysis, then cell quantity increases, but the risk of bacterial contamination increases
Solution Approach 1:
The patent applies preliminary action by establishing selectable marker systems before cell culture that enable rapid enrichment of knock-in cells. This preliminary genetic modification allows for quick identification and expansion of target cells with minimal culture time, thereby obtaining sufficient cell quantities while minimizing the window of opportunity for bacterial contamination to occur
4Reliability
If conventional knock-in methods are used, then standard procedures are followed, but productivity is low due to excessive time requirements
Solution Approach 1:
The patent uses feedback through selectable markers to rapidly identify and enrich knock-in cells. The puromycin resistance marker provides immediate feedback on successful transfection, and the subsequent FRT-flanked marker system enables rapid selection against random integrants, significantly improving productivity while maintaining knock-in accuracy through controlled selection processes
Solution Approach 2:
The patent employs intermediary elements such as the FRT site and Flp recombinase as mediators to achieve precise knock-in with improved productivity. These intermediaries enable rapid enrichment of true knock-in cells through controlled recombination events, reducing the time required while maintaining accuracy through the specificity of the recombination system
Data Source
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AI summary
The present inventors have found that the use of a site-specific nuclease system which includes a combination of a molecule that simultaneously targets and cleaves one homology arm sequence of the donor DNA and the genomic sequence corresponding to the homology arm sequence, and a molecule that targets and cleaves the genomic region in the vicinity of the cleavage site by that molecule causes repair between genomic DNA and donor DNA through both non-homologous end joining and homologous recombination, making it possible to produce cells and organisms with knocked-in long donor sequences with high efficiency and accuracy.