DNA Cleavage Detection Using 3′-Overhang Probe Hybridization
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Solution Overview
Problem
Current genome editing techniques lack effective methods for detecting whether nuclease-induced DNA cleavage is on-target or off-target, which is crucial for ensuring precision and success in genome editing outcomes.
Innovation Solution
The method involves using labeled oligonucleotides that hybridize specifically to single-strand 3′-overhangs created by nuclease cleavage, allowing detection of on-target or off-target cleavage by hybridization patterns, without denaturing the genomic DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nuclease is used for genome editing, then DNA cleavage efficiency is improved, but detection capability of on-target vs off-target cleavage deteriorates
Solution Approach 1:
The patent introduces single-strand 3'-overhangs as intermediary structures that form at nuclease cleavage sites. These overhangs serve as mediators between the cleavage event and the detection system, allowing probes to specifically bind to and reveal cleavage locations without interfering with the nuclease activity itself.
Solution Approach 2:
The patent employs detectably labeled probes that produce detectable signals (analogous to color changes) when they hybridize to the single-strand 3'-overhangs. This allows visual or instrumental detection of cleavage events, transforming the invisible molecular event into a detectable signal that distinguishes on-target from off-target cleavage.
2Measurement precision
If genomic DNA is denatured for detection, then probe hybridization sensitivity is improved, but genomic DNA structural integrity deteriorates
Solution Approach 1:
The patent extracts or isolates the single-strand 3'-overhang regions from the double-stranded genomic DNA context. By focusing detection only on these exposed single-stranded regions rather than requiring denaturation of the entire genome, the method achieves high probe hybridization sensitivity while preserving the overall structural integrity of the genomic DNA.
3Ease of operation
If detection method is simplified, then ease of operation is improved, but measurement precision deteriorates
Solution Approach 1:
The patent performs preliminary action by allowing the nuclease to naturally generate single-strand 3'-overhangs at cleavage sites before detection. This preliminary structural change occurs automatically as part of the cleavage process, eliminating the need for complex sample preparation or denaturation steps, while the subsequent probe hybridization maintains high precision in distinguishing cleavage types.
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 precise identification of on-target and off-target cleavage events, enhancing the accuracy and reliability of genome editing by distinguishing between intended and unintended DNA cuts.
Implementation Method 1
contacting the fixed cell with a plurality of oligonucleotides complementary to a single stranded (ss) 3'-overhang created at the cleavage site, where the genomic DNA is not denatured and detecting hybridization of the plurality of oligonucleotides to the ss 3'-overhang
Data Source
AI summary
Provided herein are methods and compositions for use in detecting on-target and/or off-target cleavage of genomic DNA in a cell by a nuclease. The on-target and off-target cleavage is detected by using oligonucleotides that bind to the single-stranded 3′-overhang created at the cleavage site. The nuclease may be an engineered nuclease comprising a nucleic acid binding domain that binds to a target nucleic acid of interest.