Base Editor Editing Site Detection Kit
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Solution Overview
Problem
Current methods for detecting off-target effects of base editors in gene editing are limited by low sensitivity and accuracy, particularly due to uneven sequencing coverage and interference from genomic background noise, leading to inconsistent and biased results.
Innovation Solution
A method involving the generation of a single-strand break at the edited base site, followed by labeling and enrichment of the editing intermediate using nucleotides with specific labeling molecules, allows for high-sensitivity detection of editing sites and off-target effects across the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Area of stationary object
If whole genome sequencing is used to detect off-target effects, then comprehensive coverage is achieved, but sensitivity is reduced due to uneven sequencing coverage and genomic background noise
Solution Approach 1:
The patent segments the detection process into multiple steps: first generating single-strand breaks at edited bases using specific endonucleases, then performing targeted labeling and enrichment of break sites before sequencing. This segmentation allows focused detection at editing sites rather than attempting to detect all potential off-target sites across the entire genome, thereby improving sensitivity while maintaining comprehensive coverage.
Solution Approach 2:
The patent extracts and enriches DNA fragments containing single-strand breaks at editing sites using labeling molecules and affinity purification. By taking out and concentrating these specific fragments from the complex genomic background, the method achieves high detection sensitivity even with limited sequencing depth, resolving the contradiction between comprehensive coverage and detection sensitivity.
2Quantity of substance
If conventional sequencing methods are used, then cost and data volume are reduced, but accuracy is worsened due to interference from natural variations and sequencing errors
Solution Approach 1:
The patent performs preliminary enrichment of DNA fragments at editing sites before sequencing by using endonucleases to generate single-strand breaks, followed by labeling and affinity purification. This preliminary action concentrates the target sequences of interest, allowing accurate detection with reduced sequencing data volume and minimizing the impact of natural variations and sequencing errors.
Solution Approach 2:
The patent introduces labeling molecules as intermediaries that specifically bind to single-strand break sites. These labeled intermediaries enable selective enrichment and identification of editing sites, improving detection accuracy while reducing the need for extensive sequencing data to distinguish true editing events from background noise.
3Productivity
If base editing is performed without detection method, then editing efficiency is improved, but reliability is worsened due to inability to identify off-target effects
Solution Approach 1:
The patent implements a feedback mechanism where the detection method provides information about off-target editing events, allowing researchers to evaluate and improve base editor specificity. The detection results feed back into the design and optimization of base editors, enabling continuous improvement of both editing efficiency and reliability by identifying and correcting off-target effects.
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 editing sites and off-target effects with improved sensitivity and accuracy, reducing the need for extensive sequencing data and minimizing interference from natural variations and sequencing errors.
Implementation Method 1
generating a single-strand break at the edited base site in the first nucleic acid strand
Implementation Method 2
introducing a labeled nucleotide at or downstream of the single-strand break to produce a labeled product
Implementation Method 3
isolating or enriching the labeled product by using a binding molecule capable of specifically recognizing and binding the labeling molecule
Data Source
AI summary
Provided are a method for detecting nucleic acid sites edited by a base editor, and a kit for implementing the method. Also provided is a method for detecting the editing efficiency or off-target effects of the base editor editing nucleic acids.