Allele Editing With Surface Isoform Detection for Single-Cell HDR
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for quantifying homology directed repair (HDR) events in gene editing are inefficient and require transgenes or prior manipulation of cells, limiting their application to cell lines and not primary cells, and there is a need for a simple, cost-effective system for rapid single-cell based quantification and selective depletion of edited cells.
Innovation Solution
A method involving the induction of a DNA double strand break, provision of a DNA repair construct with homology arms, and detection of surface protein isoforms to determine HDR events, allowing for rapid quantification and selective depletion of cells without transgenes, using ligands to distinguish between isoforms and employing HDR enhancing reagents like vanillin and rucaparib.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry-based reporter systems are used to quantify gene editing on a single cell basis, then measurement precision is improved, but device complexity increases due to the need for genetic manipulation of cells prior to use
Solution Approach 1:
The invention introduces allele markers (such as CD45.1 and CD45.2 isoforms) into cells in advance, before the gene editing assay is performed. This preliminary marking allows for direct detection of HDR events without requiring additional genetic manipulation during the assay, thereby maintaining single-cell measurement precision while reducing operational complexity
Solution Approach 2:
The invention uses allele-specific ligands (antibodies) as intermediaries to detect HDR events. These ligands bind to specific allele markers on the cell surface, enabling flow cytometry-based detection of edited cells without requiring direct genetic manipulation of the assay cells. The ligands serve as mediators that translate molecular-level HDR events into detectable phenotypic signals
2Measurement precision
If cloning of cell lines is performed to obtain single cell information, then measurement precision is improved, but loss of time increases due to the tedious process
Solution Approach 1:
The invention replaces the mechanical/clonal expansion process with a direct molecular detection approach. Instead of physically isolating and expanding single cells through cloning, the invention uses allele-specific ligands to directly detect and quantify HDR events in individual cells through flow cytometry, substituting a time-consuming mechanical process with a rapid biochemical detection method
3Productivity
If NHEJ pathway is exploited for gene editing, then productivity is improved, but manufacturing precision deteriorates due to random insertions and deletions
Solution Approach 1:
The invention implements a feedback mechanism where allele-specific ligands detect the outcome of HDR events in real-time, allowing for the selection and enrichment of precisely edited cells. This feedback loop enables the HDR pathway to achieve both high productivity and high precision by identifying and isolating cells with the desired genetic modifications
Solution Approach 2:
The invention changes the detection parameter from bulk population sequencing to single-cell allele-specific detection. By measuring the presence of specific allele markers at the single-cell level, the invention can distinguish between HDR and NHEJ events, enabling selective enrichment of precisely edited cells while maintaining high overall editing productivity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables efficient, single-cell based quantification of HDR events and selective depletion of edited cells, applicable to primary cells, enhancing HDR efficiency and enabling precise gene editing and tracking in vivo.
Implementation Method 1
Double strand DNA (dsDNA) breaks can be induced at desired genomic loci through the use of 'programmable', user-defined short guide RNAs which complex with a nuclease. Frequently used nucleases include Gas proteins, particularly Cas9
Implementation Method 2
The HDR pathway provides the opportunity to introduce precise mutations by repairing a (ds)DNA break based on a DNA template
Implementation Method 3
such systems depend on genetic manipulation of the assessed cells or organisms (mostly prior to their use), thus restricting their use
Data Source
AI summary
The invention relates to a method to determine a homology directed repair (HDR) event within a eukaryotic cell, wherein the cell expresses a first isoform of a surface protein, which is different from a second isoform of said surface protein with regard to an amino acid marker. The method comprises the steps of inducing a DNA double strand break, providing a HDR template DNA construct comprising the amino acid marker corresponding to the second isoform of the surface protein and subsequently determining the expression of the first or second isoform of said surface protein on said cell, wherein expression of the second isoform indicates a successful HDR event. The invention also relates to a method for editing a genomic location of interest within a eukaryotic cell, and to a method of selectively depleting or enriching an edited cell in a composition of non-edited and edited cells.