Molecular Combing for Genome Editing Detection
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Solution Overview
Problem
Current methods for detecting and quantifying genome modifications induced by modified nucleases, such as CRISPR-Cas9, are inefficient and prone to bias due to pre-analytical steps, and fail to accurately detect rare or off-target events in a single assay.
Innovation Solution
The use of Molecular Combing combined with Genomic Morse Code (GMC) allows for the direct inspection and counting of individual genome editing events without pre-analytical steps, enabling precise quantification of both expected and rare events, including on- and off-target modifications in a single assay.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods for detecting genome modifications are used, then detection can be performed, but the methods are inefficient and prone to bias due to pre-analytical steps
Solution Approach 1:
The invention extracts and eliminates the problematic pre-analytical steps from the detection workflow. By using molecular combing to directly visualize and count individual genome editing events without requiring PCR amplification, cloning, or sequencing, the method removes the sources of bias and inefficiency while maintaining detection accuracy.
Solution Approach 2:
The invention replaces complex biochemical analysis systems (PCR, cloning, sequencing) with a direct optical visualization system using molecular combing and fluorescence microscopy. This substitution eliminates the need for multiple pre-analytical steps while providing accurate detection and quantification of genome editing events.
2Reliability
If current methods are used, then genome editing events can be detected, but rare or off-target events cannot be accurately detected in a single assay
Solution Approach 1:
The molecular combing method provides universal detection capability that can identify all types of genome editing events (on-target and off-target) within a single assay. By directly visualizing individual DNA molecules and their modification events, the system achieves both high reliability for detecting expected events and broad adaptability for detecting rare or unexpected modifications.
3Measurement precision
If Molecular Combing with Genomic Morse Code is used, then precise quantification of genome editing events is enabled, but the method requires direct inspection and counting of individual events
Solution Approach 1:
The invention uses fluorescently labeled probes that hybridize to specific genomic sequences, creating visible color signals that can be directly observed and counted using fluorescence microscopy. The Genomic Morse Code approach uses patterns of fluorescent signals along stretched DNA molecules to encode and reveal modification events, enabling precise quantification through direct visual inspection without requiring complex analytical procedures.
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 provides a more efficient and accurate method for detecting and quantifying genome editing events, avoiding bias and enabling the detection of rare modifications that were previously undetectable, thereby improving the quality control of editing procedures.
Implementation Method 1
Molecular combing is a technique enabling the direct visualization of individual nucleic acid molecules... After stretching, it is possible to hybridize sequence-specific probes
Implementation Method 2
After stretching, it is possible to hybridize sequence-specific probes detectable for example by fluorescence microscopy
Implementation Method 3
hybridize sequence-specific probes detectable for example by fluorescence microscopy... The length of the fluorescent signals and/or their number, and their spacing on the slide provides a direct reading
Data Source
AI summary
Methods for detecting and characterizing large genomic rearrangements induced by modified nucleases at high resolution and for quantifying the frequency of the large genomic or gene rearrangements induced by modified nucleases using Molecular Combing.