Inducible gRNA Singulation for Editing Efficiency
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Solution Overview
Problem
Current nucleic acid-guided nuclease gene editing methods face challenges in efficiently identifying and enriching edited cells due to rapid depletion of edited cell types and selective enrichment of unedited cells, particularly in pooled or multiplex formats, where constitutive expression of editing components leads to growth bias and reduced viability.
Innovation Solution
The implementation of tightly-regulated expression of nucleic acid-guided nuclease editing system components using inducible guide RNA constructs, combined with singulation and cherry picking techniques, allows for controlled editing and enrichment of edited cells by separating transformation and editing processes, and enabling high-throughput screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If constitutive expression of nuclease components is used to drive high efficiency editing, then editing efficiency is improved, but edited cell types are rapidly depleted and unedited cells are selectively enriched
Solution Approach 1:
The patent transforms cells with editing components (nuclease and gRNA) in advance, then delays nuclease activation until after singulation. This preliminary transformation without immediate editing allows cells to be prepared and separated before the editing action occurs, preventing early depletion of edited cells during the transformation phase.
Solution Approach 2:
The patent implements a two-phase process: Phase 1 involves transformation and singulation without active editing, Phase 2 involves inducing nuclease expression after singulation. This periodic activation of editing—off during transformation/singulation, on during enrichment—resolves the contradiction by separating the harmful depletion effect from the desired editing effect.
2Productivity
If constitutive expression of editing components is used in pooled or multiplex formats, then high throughput editing is achieved, but growth bias occurs and edited cell viability is reduced
Solution Approach 1:
The patent segments the editing process into distinct phases: transformation phase (with components but no active editing), singulation phase (physical separation), and editing phase (induced nuclease expression). This segmentation isolates the harmful growth bias effects to specific phases while maintaining high throughput capability through parallel processing of multiple cell types.
Solution Approach 2:
The patent introduces singulation as an intermediary step between transformation and editing. This intermediate physical separation process allows cells to be prepared and divided into individual cultures before editing occurs, eliminating competitive growth interactions that cause bias and stress in pooled formats.
3Loss of time
If early editing is performed after transformation, then editing occurs in pooled format, but transformed cells are not yet acclimated and viability is compromised
Solution Approach 1:
The patent performs preliminary transformation and acclimation of cells to the editing components before initiating nuclease expression. Cells are given time to recover and adapt to the presence of gRNA and other components without suffering the cytotoxic effects of active DNA cleavage, thereby improving viability while maintaining efficient editing timing.
Data Source
AI summary
The present disclosure provides instruments, modules and methods for improved detection of edited cells following nucleic acid-guided nuclease genome editing. The disclosure provides improved automated instruments that perform methods—including high throughput methods—for screening cells that have been subjected to editing and identifying cells that have been properly edited.