Endonuclease Barcoding for High-Throughput Cell Tracking
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Solution Overview
Problem
Current DNA editing tools, such as CRISPR/Cas9, face challenges including flexibility issues, off-target DNA cleavage, variable efficiency, and incompatibility with cell growth modifications, requiring time-consuming clone selection and limiting high-throughput applications in eukaryotic cells.
Innovation Solution
A method involving endonuclease-based 'barcoding' where silent mutations are introduced to label cells, allowing for the detection of genetic modifications by comparing distinct signatures within cell populations, reducing the need for clone selection and enhancing high-throughput capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If endonuclease-based genome editing is used to introduce precise genetic modifications, then manufacturing precision is improved, but loss of time increases due to required clone selection
Solution Approach 1:
The patent applies preliminary action by introducing barcoding sequences and silent mutations during the initial genome editing step. These barcodes are incorporated into the donor DNA template before the editing process, so that when HDR occurs, the barcode is simultaneously introduced. This preliminary preparation eliminates the need for subsequent clone selection and sequencing to identify edited cells, as the barcode provides immediate identification.
Solution Approach 2:
The patent uses copying by creating unique barcode sequences that are copied into the genome along with the desired genetic modification. These barcodes serve as molecular copies or tags that can be detected and used to identify and track specific edited cells without requiring further manipulation or selection steps.
2Adaptability or versatility
If conventional DNA editing tools are used, then adaptability is improved, but object-generated harmful factors worsen due to off-target DNA cleavage
Solution Approach 1:
The patent implements feedback by using the introduced barcodes to monitor and identify cells that have successfully undergone the desired editing. The barcode system provides a readout mechanism that confirms on-target editing occurred, allowing researchers to select only those cells with the intended modifications. This feedback loop enables verification of editing specificity without requiring extensive off-target analysis.
Solution Approach 2:
The barcode sequences serve as intermediaries between the genome editing process and the detection/selection system. Rather than directly detecting the genetic modification itself, the system uses the barcode as a mediator that can be easily read and used to identify edited cells. This intermediary approach simplifies verification and reduces the need for complex off-target screening.
3Ease of operation
If CRISPR/Cas9 system is used for genome editing, then ease of operation is improved, but reliability worsens due to variable editing efficiency
Solution Approach 1:
The barcode system provides feedback that allows researchers to determine the actual editing efficiency of their CRISPR/Cas9 experiment. By sequencing or detecting the barcodes in the cell population, researchers can quantify what fraction of cells successfully underwent HDR with the donor template. This feedback enables optimization of editing conditions and accurate assessment of experimental outcomes.
Solution Approach 2:
The system applies self-service by allowing the edited cells to carry their own identification tags (barcodes) that enable automatic identification and tracking. The barcodes are self-incorporated during the HDR process, eliminating the need for separate labeling steps or complex selection procedures, thereby maintaining ease of operation while improving reliability through accurate efficiency measurement.
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
This approach enables fast, convenient, and effective characterization of genetic modifications in eukaryotic cells, reducing off-target effects and improving the efficiency of DNA editing by allowing for the tracking of modified cells within heterogeneous populations.
Implementation Method 1
The mechanism behind endonuclease-based genome editing generally requires a first step of DNA single or double strand break
Implementation Method 2
In HDR, a donor nucleic acid co-introduced into the cells functions as a template for precise repair
Implementation Method 3
the error-prone nonhomologous end-joining (NHEJ)
Data Source
AI summary
The instant invention relates to a method for labeling endonuclease-treated cells, comprising the steps of a) providing cells or a composition comprising cells; b) bringing into contact said cells or said composition with: —at least one endonuclease suitable for targeting a genomic region of interest in said cells, or a vector suitable for expressing said endonuclease in said cells; —at least one first nucleic acid suitable for introducing one or more silent mutation(s) in said genomic region by homology-directed repair (HDR), and optionally one or more non-silent mutation(s); and —at least one second nucleic acid suitable for introducing one or more silent mutation(s) in said genomic region by homology-directed repair (HDR), but distinct from the silent mutations of the first nucleic acid; thereby labeling endonuclease-treated cells.