Inducible Guide RNA Constructs 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 biases.
Innovation Solution
The development of instruments and methods that enable automated high-throughput screening and enrichment of edited cells through isolation or substantial isolation, normalization, and cherry picking, utilizing inducible guide RNA constructs and solid wall devices to separate and select edited cells based on growth characteristics.
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 employs periodic induction of nuclease expression using doxycycline pulses rather than constitutive expression. Cells are exposed to doxycycline for specific time periods (e.g., 24-48 hours) to induce nuclease activity, then the induction is removed to allow unedited cells to recover and continue dividing. This periodic action maintains editing efficiency while preventing complete depletion of edited cell types and selective enrichment of unedited cells.
Solution Approach 2:
The system transitions from static constitutive expression to dynamic inducible expression. The nuclease expression level can be adjusted in real-time based on experimental needs, allowing optimization of the balance between editing efficiency and cell population stability. The inducible promoter system enables flexible control over when and how strongly the nuclease is expressed.
2Productivity
If pooled or multiplex formats are used for editing, then throughput is improved, but growth bias from unedited cells occurs leading to selective enrichment
Solution Approach 1:
The patent performs preliminary isolation of individual cells into separate wells before editing occurs. This pre-separation prevents growth bias from affecting the editing process itself, as each cell is contained individually. After editing and a period of growth, colonies are normalized to equal sizes, and then pooled for analysis. This preliminary action maintains high throughput while eliminating growth bias interference.
Solution Approach 2:
The cell population is segmented into individual cells in separate wells during the editing phase, allowing independent processing of each cell. This segmentation prevents competitive growth interactions that would otherwise cause selective enrichment of unedited cells. After editing completion, the segmented colonies are recombined into a pooled format for high-throughput analysis.
3Measurement precision
If isolation or substantial isolation is performed followed by growth normalization, then editing efficiency observation is improved up to 4-fold, but device complexity increases
Solution Approach 1:
The solid wall isolation device utilizes the cells' own growth characteristics to achieve normalization. After isolation and editing, colonies are allowed to grow to fill the available space in each well. The physical constraint of the well walls provides automatic normalization without requiring external intervention or complex control mechanisms. The device structure itself enables the self-normalization process.
Solution Approach 2:
The solid wall device acts as an intermediary structure that facilitates both isolation and normalization functions. The microwell array provides physical separation for individual cell isolation while simultaneously providing standardized growth chambers that enable automatic normalization. This intermediary structure combines multiple functions into a single device, reducing overall system complexity.
4Measurement precision
If cherry picking of edited cell colonies is implemented, then observed editing efficiency increases up to 8-fold, but automation difficulty increases
Solution Approach 1:
The patent employs fluorescent reporters that cause edited colonies to exhibit distinct optical properties compared to unedited colonies. This optical differentiation enables automated detection systems to identify and select edited colonies based on their fluorescence characteristics. The color/fluorescence change provides an easily detectable signal that simplifies automation of the cherry-picking process.
Solution Approach 2:
The manual mechanical process of cherry-picking colonies is replaced with automated optical detection and robotic manipulation systems. Fluorescence-activated sorting or automated imaging systems identify edited colonies based on their optical properties, and robotic arms or liquid handling systems automatically transfer or harvest the selected colonies. This substitution eliminates manual intervention while maintaining high precision in editing efficiency measurement.
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.