Genome Editing Break Quantitation via Donor Insertion and Fragment Analysis
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Solution Overview
Problem
Current genome editing reagents often introduce double-stranded breaks at both on-target and off-target sites in the genome, making it difficult to predict and quantify these breaks, especially in populations of cells, and existing methods for identification and quantitation are costly and time-consuming.
Innovation Solution
A method involving the insertion of an exogenous donor polynucleotide into double-stranded break points, followed by fragmentation, partitioning, and selective amplification using complementary primers to quantify the number of double-stranded breaks, allowing for the detection of on-target and off-target editing sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome next generation sequencing is used to identify and quantitate double-stranded breaks, then measurement precision is improved, but cost and time consumption increase significantly
Solution Approach 1:
The genome is fragmented into smaller pieces, and double-stranded breaks are detected in individual fragments rather than sequencing the entire genome. This segmentation allows focused detection of break points without the need for comprehensive whole-genome sequencing, reducing time and cost while maintaining precision in identifying break locations.
Solution Approach 2:
The method extracts and isolates specific genomic fragments containing double-stranded breaks from the complex genomic material. By using selective amplification and detection approaches, the technique extracts only the relevant information about break points without requiring analysis of the entire genome, thereby reducing time consumption while preserving measurement precision.
2Measurement precision
If whole genome next generation sequencing is used to quantitate double-stranded breaks, then measurement precision is improved, but cost increases significantly
Solution Approach 1:
The method uses inexpensive, disposable genomic DNA fragments as the basis for detection. Instead of investing in expensive whole-genome sequencing, the approach uses affordable PCR amplification and detection of specific fragments containing breaks. This substitution of expensive sequencing with cheaper fragment-based analysis reduces cost while maintaining quantitation precision.
Solution Approach 2:
The method creates multiple copies of specific genomic fragments containing double-stranded breaks through PCR amplification. These amplified copies serve as proxies for the original break-containing sequences, allowing precise quantitation without the need for expensive sequencing. The copying process generates sufficient material for accurate measurement at a fraction of the cost of whole-genome sequencing.
3Productivity
If genome editing reagents are used to create double-stranded breaks, then productivity is improved, but off-target breaks occur at unpredictable sites
Solution Approach 1:
The method performs preliminary detection of off-target double-stranded breaks before final genome editing outcomes are assessed. By using the described fragment-based detection approach, researchers can identify unintended break sites in advance, allowing them to evaluate the full spectrum of editing events including off-target effects, thereby improving reliability while maintaining productivity.
Solution Approach 2:
The detection method provides feedback information about both on-target and off-target double-stranded breaks. By quantifying breaks at expected target sites versus unexpected off-target sites, the method generates feedback that allows optimization of genome editing reagents to improve specificity. This feedback loop enables continuous improvement of reliability without sacrificing productivity.
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 method enables efficient and cost-effective quantitation of double-stranded breaks in cells, distinguishing between on-target and off-target edits, facilitating the optimization of genome editing reagents and assessing genetic damage from mutagens or radiation.
Implementation Method 1
the pair of amplification primers are complementary to opposite strands of the donor polynucleotide or portion thereof, and wherein the pair of amplification primers are oriented such that the 5' ends are proximal to each other and the 3' ends are distal to each other when hybridized to a genomic nucleic fragment containing the inserted donor polynucleotide
Implementation Method 2
amplifying the genomic nucleic fragments with the pair of amplification primers to selectively generate amplicons in mixture partitions that contain one or more genomic fragments containing the inserted donor polynucleotide
Data Source
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AI summary
Methods, compositions, and kits are provided for quantifying a number or frequency of double stranded breaks in the genome of a cell or in the genomes of a population of cells.