Automated Nuclease Editing Enrichment via Cell Cycle Synchronization
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Solution Overview
Problem
Current methods for nucleic acid-guided nuclease gene editing face challenges in efficiently generating edits in cell populations and enriching for edited cells due to selective enrichment of unedited cells and growth biases resulting from double-strand DNA breaks and differential editing rates.
Innovation Solution
The implementation of an automated high-throughput enrichment method that induces nucleic acid-guided nuclease editing at specific points in the cell growth cycle, using an inducible promoter to control the nuclease and gRNA, allowing cells to grow until they reach the stationary phase before editing, thereby overcoming growth biases and improving editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nucleic acid-guided nuclease editing is performed in conventional cell culture methods, then editing can be achieved, but unedited cells are selectively enriched due to lack of double-strand DNA breaks, reducing editing efficiency
Solution Approach 1:
The patent applies preliminary action by inducing nuclease expression before the editing step to create a pool of cells with varying editing statuses, then using flow cytometry to detect and sort edited cells based on DNA damage markers. This preliminary induction allows subsequent enrichment of truly edited cells while eliminating unedited cells that would otherwise be selectively enriched during conventional culture.
2Productivity
If editing is performed during active cell growth, then cell proliferation occurs, but growth biases and fitness effects skew the representation of different edits in the population
Solution Approach 1:
The patent employs periodic action by synchronizing cell cultures to specific phases of the cell cycle before inducing nuclease expression. Cells are allowed to progress through defined growth phases, then editing is induced at a specific point, followed by detection and sorting. This periodic synchronization eliminates continuous growth biases while maintaining sufficient cell population expansion for accurate edit frequency measurement.
3Manufacturing precision
If double-strand DNA breaks are introduced during editing, then genome editing occurs, but cell viability is dramatically negatively impacted
Solution Approach 1:
The patent uses flow cytometry detection of DNA damage markers as an intermediary to identify cells that have successfully undergone editing without requiring them to survive prolonged culture. By detecting edited cells immediately after editing based on DNA damage signatures, the method captures editing events before viability loss eliminates them from the population, thus preserving both editing precision and cell viability information.
Data Source
AI summary
The present disclosure provides modules, instruments and methods to enrich for cells edited via nucleic acid-guided nuclease editing of live cells.