Nucleic Acid Target Enrichment Using CRISPR Overhang Labeling
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Solution Overview
Problem
Existing methods for enriching nucleic acid target sequences, such as circulating tumor DNA, suffer from poor coverage in regions of interest and fail to effectively identify trace amounts due to low frequency in nucleic acid samples.
Innovation Solution
A method involving cutting nucleic acid molecules with CRISPR-Cas nucleases to create single-stranded overhangs, filling them with labeled nucleotides, and capturing the molecules using capture domains like streptavidin beads to enrich target sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If reduced-representation bisulfite sequencing, whole-genome bisulfite sequencing, or methylated DNA immunoprecipitation sequencing approaches are used to enrich methylated DNA sequences, then genome-wide information is available, but coverage in regions of interest is poor
Solution Approach 1:
The patent segments the nucleic acid molecules by cutting them at specific locations to generate single-stranded overhangs. This segmentation allows selective enrichment of target sequences while maintaining genome-wide information, resolving the contradiction between comprehensive coverage and regional depth.
Solution Approach 2:
The patent applies local quality by filling in overhangs with labeled nucleotides at specific cut sites. This creates localized labels that enable selective capture of target sequences in regions of interest while preserving overall genome-wide coverage through the labeling approach.
2Quantity of substance
If target sequences are present at low frequency in nucleic acid samples, then detection becomes challenging, but amplification and sequencing may fail
Solution Approach 1:
The patent performs preliminary action by cutting nucleic acid molecules and filling in overhangs with labeled nucleotides before sequencing. This preliminary enrichment step increases the frequency of target sequences in the sample, making them detectable even when originally present at low frequency, thereby improving detection reliability.
Solution Approach 2:
The patent uses labeled nucleotides as intermediaries to bridge the gap between low-frequency target sequences and detection systems. The labels serve as mediators that enable selective capture and amplification of target sequences, transforming low-frequency targets into detectable signals.
3Measurement precision
If nucleic acid molecules are cut to form single-stranded overhangs and filled with labels, then target sequence enrichment is improved, but process complexity increases
Solution Approach 1:
The patent replaces complex mechanical separation systems with biochemical mechanisms. Instead of using complex physical separation devices, the method uses enzymatic cutting and label filling followed by selective capture, simplifying the overall process while achieving effective enrichment.
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
Enhances the enrichment of target sequences, allowing for improved detection and sequencing of low-frequency nucleic acid targets by increasing their representation in nucleic acid libraries.
Implementation Method 1
cutting a nucleic acid molecule that includes a target sequence to generate a single stranded overhang at a cut end of the molecule
Implementation Method 2
filling in the overhang with at least one labeled nucleotide
Implementation Method 3
enriching the molecule that includes the target by contacting at least one of the labeled nucleotides in the molecule with a capture domain
Data Source
AI summary
The invention provides methods for enriching nucleic acid target sequences from a sample, for example, from a biological sample or from a nucleic acid library.