Microfluidic Sequestration for Precise Genome Editing
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Solution Overview
Problem
Current genome modification technologies face challenges with non-homologous end-joining (NHEJ) errors and off-target modifications, which are costly and time-consuming to identify and correct.
Innovation Solution
A method using a microfluidic device with sequestration pens to generate clonal populations of genetically modified cells, involving genome editing biomolecules and donor template nucleic acids to achieve precise genome edits, with detection of on-target and off-target edits through nucleic acid sequencing and amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If targeted nucleases are used for genome editing, then target specificity is improved, but off-target modifications still occur
Solution Approach 1:
The patent employs a two-stage verification process where initial genome editing is followed by sequential detection methods (first detection method and second detection method) to identify and filter off-target modifications. This feedback mechanism ensures that only cells with desired on-target edits and without off-target effects are selected for clonal expansion.
Solution Approach 2:
The patent replaces traditional mechanical/manual screening methods with automated detection systems that use molecular biology techniques (such as PCR, sequencing, or other nucleic acid detection methods) to automatically identify cells with correct genome edits, thereby improving precision while reducing human error and time consumption.
2Measurement precision
If comprehensive detection of genome edits is performed, then accuracy is improved, but time and cost increase
Solution Approach 1:
The patent performs preliminary enrichment of candidate cells based on initial screening criteria before conducting comprehensive verification. This preliminary action reduces the number of cells requiring full verification, thereby maintaining high detection accuracy while reducing overall time and resource consumption.
Solution Approach 2:
The detection process is divided into multiple independent stages (first detection method, second detection method, clonal expansion verification) that can be performed sequentially on different cell subsets. This segmentation allows comprehensive detection to be distributed across multiple steps rather than requiring all cells to undergo all detection methods simultaneously, reducing total detection time.
3Stability of the object's composition
If clonal populations are expanded from single cells, then genetic homogeneity is improved, but risk of propagating off-target edits increases
Solution Approach 1:
The patent performs comprehensive genome verification on single parent cells before initiating clonal expansion. This preliminary verification ensures that only cells confirmed to have correct on-target edits and free of off-target modifications are used as founders for clonal populations, thereby ensuring genetic homogeneity without propagating harmful off-target edits.
Solution Approach 2:
The patent implements a feedback verification system where clonal populations are monitored during and after expansion to ensure they maintain the desired genetic characteristics. This continuous feedback allows early detection and correction of any deviations, ensuring that genetic homogeneity is maintained without propagating off-target edits.
Data Source
AI summary
Methods are described herein for isolating clonal populations of cells having a defined genetic modification. The methods are performed, at least in part, in a microfluidic device comprising one or more sequestration pens. The methods include the steps of: maintaining individual cells (or precursors thereof) that have undergone a genomic editing process in corresponding sequestration pens of a microfluidic device; expanding the individual cells into respective clonal populations of cells; and detecting, in one or more cells of each clonal population, the presence of a first nucleic acid sequence that is indicative of the presence of an on-target genome edit in the clonal population of cells. Also described are methods of performing genome editing within a microfluidic device, and compositions comprising one or more clonal populations of cells generated according to the methods disclosed herein.