Optical Mapping for Quantifying Gene Editing Efficiency
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Solution Overview
Problem
Current methods for analyzing genome editing, such as whole genome sequencing, are inefficient and costly, particularly for detecting off-target editing, as they require sequencing of entire genomes and are not effective at low editing efficiencies.
Innovation Solution
A method involving optical-based genome mapping, where genomic DNA is motif-labeled with a first fluorescent tag at known locations to create an optical map, and then edited with nucleotides containing a second fluorescent tag. This allows for the detection and quantification of genome editing efficiencies by identifying the locations of the second tag relative to the first tag.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If whole genome sequencing is used for off-target analysis, then unbiased survey of the full genome is achieved, but throughput is limited and cost increases
Solution Approach 1:
The patent segments the genome analysis process by first creating an optical map of the entire genome, then selectively analyzing only the regions where editing events occur. This divides the comprehensive WGS approach into targeted analysis of specific loci, improving throughput while maintaining detection accuracy.
Solution Approach 2:
The patent extracts and analyzes only the relevant portions of the genome that contain editing events, rather than sequencing the entire genome. By isolating and examining only the modified regions identified through optical mapping, the method achieves high throughput without sacrificing off-target detection capability.
2Measurement precision
If whole genome sequencing is used for off-target analysis, then comprehensive genome coverage is achieved, but cost increases
Solution Approach 1:
The patent segments the expensive WGS step into a preliminary optical mapping step followed by targeted analysis. This reduces the quantity of sequencing required, thereby lowering cost while maintaining the ability to detect off-target events with high accuracy.
Solution Approach 2:
The patent extracts only the necessary information from the genome by using optical mapping to identify editing events, then focuses sequencing resources only on those specific regions. This extraction approach dramatically reduces the amount of sequencing needed, lowering cost while preserving detection accuracy.
3Measurement precision
If detection sensitivity is increased to identify low efficiency off-target editing, then detection capability improves, but WGS becomes impractical and costly
Solution Approach 1:
The patent segments the detection process into two stages: optical mapping for sensitive detection of all editing events across the genome, followed by targeted analysis of identified regions. This segmentation enables high detection sensitivity without the impracticality of comprehensive WGS.
Solution Approach 2:
The patent extracts editing event locations through optical mapping with high sensitivity, then extracts and analyzes only those specific regions. This two-stage extraction approach maintains high detection sensitivity for low efficiency off-target events while keeping the method practical and cost-effective.
4Measurement precision
If targeted sequencing with PCR amplification is used for on-target analysis, then specific gene analysis is achieved, but quantification of editing efficiency becomes difficult and biased
Solution Approach 1:
The patent replaces the mechanical PCR amplification process with optical mapping technology. This substitution eliminates the biases introduced by PCR amplification and enables accurate, unbiased quantification of editing efficiency while maintaining the ability to analyze specific genes.
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 the detection and quantification of both on-target and off-target genome editing with high sensitivity, capable of identifying 1% of gene modification events, and is more efficient and cost-effective than traditional whole genome sequencing.
Implementation Method 1
motif-labeled with a first fluorescent tag at known locations to create an optical map
Implementation Method 2
edited with nucleotides containing a second fluorescent tag. This allows for the detection and quantification of genome editing efficiencies by identifying the locations of the second tag
Data Source
AI summary
Described herein are methods useful for quantification of on-target and off-target gene editing activity. The method comprises establishing a first consensus labeling in a genomic DNA in a first plurality of cells, which comprises introducing a first tag at a first plurality of pre-determined locations in the genomic DNA; performing the genomic editing assay with a nucleotide comprising a second tag; identifying locations of genome editing activities by detecting signals of the second tag in reference to signals of the first tag in the first consensus labeling; and calculating genome editing efficiencies at an on-target location or an off-target location.