CHANGE-seq Genome Editing Nuclease Activity Characterization
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Solution Overview
Problem
Current methods for evaluating the genome-wide activity of genome editing nucleases are labor-intensive, require large DNA inputs, and have limitations such as high processing times and the need for specialized equipment, making it difficult to comprehensively assess the safety and specificity of genome editing therapies.
Innovation Solution
The development of CHANGE-seq, a high-throughput method that involves tagmentation, gap-repairing, and intramolecular circularization of DNA using a transposome complex and ligase, followed by exonuclease treatment to produce a library of covalently closed circular DNA molecules, allowing for efficient characterization of genome-wide editing activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (GUIDE-seq, CIRCLE-seq) are used to evaluate genome-wide activity of editing nucleases, then measurement precision and reliability are improved, but loss of time and productivity deteriorate due to labor-intensive processing
Solution Approach 1:
The method segments the genome-wide analysis into targeted deep sequencing of predicted off-target sites, allowing focused high-throughput evaluation without requiring comprehensive genome-wide sequencing, thus reducing processing time while maintaining detection sensitivity
Solution Approach 2:
The method performs preliminary in silico prediction of off-target sites before experimental validation, allowing pre-selection of candidate sites for deep sequencing and reducing the scope of experimental work required, thereby decreasing processing time while preserving measurement precision
2Measurement precision
If conventional methods are used, then measurement precision is improved, but device complexity and ease of operation worsen due to requirement for specialized equipment
Solution Approach 1:
The method uses universal next-generation sequencing technology that can be applied to multiple genome editing validation needs, eliminating the requirement for specialized equipment and making the approach accessible to standard molecular biology laboratories while maintaining high detection sensitivity
3Measurement precision
If conventional methods are used, then measurement precision is improved, but quantity of substance increases due to large DNA input requirements
Solution Approach 1:
The method segments the sequencing effort into targeted deep sequencing of specific predicted off-target sites rather than requiring sequencing of the entire genome, allowing high detection sensitivity to be achieved with substantially reduced DNA input quantities
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
CHANGE-seq reduces DNA input requirements, processing time, and eliminates the need for specialized equipment, enabling more comprehensive and scalable assessment of genome editing nucleases, thereby improving the evaluation of their safety and specificity.
Implementation Method 1
performing a tagmentation of the dsDNA by incubating the dsDNA with a transposome complex comprising a transposase and a transposon DNA to add sequences that enable circularization
Implementation Method 2
incubating the DNA molecules obtained in step (d) with a ligase to induce intramolecular ligation
Implementation Method 3
treating circularized DNA molecules obtained in step (e) with exonuclease(s) to produce a library of covalently closed circular dsDNA molecules
Data Source
AI summary
The invention relates to a high-throughput method for characterizing the genome-wide activity of editing nucleases in vitro.