getPCR Wild-Type DNA Quantification for Genome Editing Efficiency
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Solution Overview
Problem
Current methods for evaluating genome editing efficiency, such as DNA sequencing and mismatch-specific nuclease-based approaches, are time-consuming, costly, and laborious, and often fail to accurately detect single nucleotide mutations and large fragment deletions, introducing sequence- and length-dependent biases.
Innovation Solution
The getPCR method uses Taq polymerase to selectively amplify wild-type DNA, allowing for the quantitative determination of wild-type DNA proportion and indirect assessment of indel occurrence, thereby evaluating genome editing efficiency with improved accuracy and simplicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA sequencing or mismatch-specific nuclease methods are used to evaluate genome editing efficiency, then detection capability is provided, but the process becomes time-consuming, costly, and laborious
Solution Approach 1:
The invention extracts and utilizes the selective amplification capability of Taq polymerase for wild-type DNA sequences. By designing primers that specifically bind to wild-type sequences, the method isolates and quantifies only the unedited DNA portion, thereby indirectly measuring editing efficiency without requiring full sequencing or complex nuclease assays
Solution Approach 2:
The invention introduces Taq polymerase as an intermediary element that mediates the detection process. The polymerase's inherent selectivity for wild-type templates serves as the detection mechanism, replacing time-consuming sequencing or nuclease-based methods with a straightforward quantitative PCR approach
2Measurement precision
If PCR amplification is used to quantify editing efficiency, then quantification is achieved, but sequence- and length-dependent biases are introduced
Solution Approach 1:
Instead of directly detecting and quantifying the edited DNA sequences (which would be subject to various biases), the invention inverts the approach by detecting and quantifying the wild-type (unedited) DNA. Since the wild-type sequence is known and constant, its quantification provides an indirect but more reliable measure of editing efficiency, avoiding the biases associated with detecting diverse edited variants
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
getPCR provides a rapid, cost-effective, and accurate method for assessing genome editing efficiency, applicable to various gene editing technologies, including CRISPR/Cas9, without the need for specialized equipment, and enables precise detection of indel frequencies and single-cell clone genotyping.
Implementation Method 1
The getPCR utilizes the selective amplification characteristic of Taq polymerase in amplifying the wild-type DNA in the genome DNA to be tested to determine the proportion of the wild-type DNA by quantitatively amplifying the wild-type DNA in the amplification product
Data Source
AI summary
A method and a kit for detecting genome editing and application thereof belongs to the field of genome editing efficiency detection, and the getPCR method for determining genome editing efficiency includes quantifying wild-type DNA in a genome to be tested and calculating the percentage of the wild-type DNA to determine the genome editing efficiency. The method has been proved to have good detection accuracy and simple operation, and can be applied to all genome editing methods to quantify genome editing efficiency and screen single-cell clones.