Automated Microfluidic Droplet Isolation for Edited Cell Enrichment
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Solution Overview
Problem
Current methods for nucleic acid-guided nuclease gene editing face challenges in enriching and identifying edited cells due to selective enrichment of unedited cells and growth biases in bulk cultures, leading to low editing efficiency and difficulty in distinguishing edited cells from unedited ones.
Innovation Solution
The development of automated high-throughput methods and instruments that utilize isolation and cherry picking techniques to separate cells into clonal colonies, allowing for normalization and direct selection of edited cells by exploiting growth rate differences, thereby enhancing editing efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If bulk culture methods are used for gene editing, then cell population growth is maintained, but edited cells cannot be distinguished from unedited cells due to selective enrichment of unedited cells
Solution Approach 1:
The patent segments the bulk cell population into individual isolated cells using microfluidic droplet generation. Each cell is encapsulated in a separate droplet, preventing selective enrichment and allowing independent growth. This segmentation enables accurate measurement of editing efficiency by eliminating the competitive advantage unedited cells have in bulk culture.
Solution Approach 2:
The patent transitions from two-dimensional bulk culture to three-dimensional droplet encapsulation with spatial isolation. By adding the dimension of physical separation through droplet compartmentalization, the system enables simultaneous maintenance of cell population growth and precise distinction between edited and unedited cells through colony morphology analysis.
2Measurement precision
If isolation methods are used to separate cells, then edited cells can be identified, but the process complexity increases
Solution Approach 1:
The patent employs self-service isolation where cells automatically sort themselves into droplets based on their intrinsic properties. The microfluidic system uses flow focusing and interfacial tension to spontaneously encapsulate individual cells without requiring external manipulation or complex mechanical isolation devices, thereby reducing overall system complexity.
Solution Approach 2:
The patent replaces complex mechanical isolation systems with a fluidic-based microfluidic approach. Instead of using mechanical separators, filters, or manual isolation techniques, the system uses controlled fluid flow, surface tension, and droplet generation to achieve cell isolation, significantly simplifying the device architecture.
3Productivity
If conventional screening methods are used, then unedited cells are enriched, but editing efficiency measurement is compromised
Solution Approach 1:
The patent performs preliminary action by isolating individual cells into droplets before the gene editing process occurs. This pre-isolation prevents the subsequent selective enrichment of unedited cells that would otherwise occur during culture growth, ensuring that when cells are later screened, the measured editing efficiency accurately reflects the actual editing outcomes rather than being skewed by differential growth rates.
Data Source
AI summary
The present disclosure provides automated modules and instruments for improved detection of nuclease genome editing of live cells. The disclosure provides improved modules—including high throughput modules—for screening cells that have been subjected to editing and identifying and selecting cells that have been properly edited.