CRISPR-RCA eccDNA Detection with Dual Signal Amplification
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Solution Overview
Problem
Current eccDNA detection methods, such as circular DNA sequencing, are lengthy, costly, and lack specificity, making rapid and targeted cancer diagnosis challenging.
Innovation Solution
A rapid eccDNA detection method using CRISPR-RCA technology that amplifies target circular DNA without digesting linear DNA, employing Cas9n-sgRNA binding, single-stranded DNA probes, rolling circle amplification, and Cas14a trans-cleavage to generate a fluorescence signal.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If circular DNA sequencing method is used for eccDNA detection, then abundant circular DNA sequence information can be acquired, but the analysis cycle becomes long (1.5-2 months) and cost becomes high
Solution Approach 1:
The patent extracts only the essential information needed for detection by using CRISPR-Cas9 to specifically target and bind to unique sequences on eccDNA, rather than sequencing all circular DNA. This extraction approach retrieves sufficient diagnostic information without the time-consuming full sequencing process, reducing the analysis cycle from 1.5-2 months to a much shorter duration.
Solution Approach 2:
The patent replaces the mechanical sequencing system with a biochemical detection system based on CRISPR-Cas9 binding and rolling circle amplification. This substitution eliminates the need for complex sequencing instruments and lengthy data processing, achieving rapid detection while maintaining sufficient information for diagnostic purposes.
2Loss of information
If circular DNA sequencing method is used for eccDNA detection, then comprehensive sequence data is obtained, but the detection cost becomes high due to sequencing instruments and long-term procedures
Solution Approach 1:
The patent uses inexpensive reagents and consumables such as CRISPR-Cas9 components, rolling circle amplification primers, and fluorescent probes instead of expensive sequencing instruments. These disposable reagents enable cost-effective detection, eliminating the need for costly sequencing equipment while maintaining detection accuracy.
Solution Approach 2:
By extracting only the necessary diagnostic information through specific sequence targeting rather than comprehensive sequencing, the patent reduces material and operational costs. This selective extraction approach minimizes reagent consumption and eliminates expensive sequencing instrument requirements.
3Loss of information
If circular DNA sequencing method is used for eccDNA detection, then all circular DNA is analyzed, but the detection specificity becomes weak since only a small fraction of sequencing data is utilized
Solution Approach 1:
The patent applies local quality by targeting specific unique sequences on eccDNA using CRISPR-Cas9 guide RNAs designed to bind to particular diagnostic regions. This localized approach focuses detection on the most informative sequences rather than uniformly analyzing all circular DNA, thereby enhancing detection specificity while reducing unnecessary data processing.
Solution Approach 2:
The patent extracts and analyzes only the specific sequence information relevant to diagnosis by using targeted CRISPR binding, rather than processing all sequencing data. This selective extraction improves measurement precision by concentrating on the most diagnostic fragments, eliminating the waste of analyzing non-informative sequences.
4Loss of time
If CRISPR-RCA technology is used for rapid eccDNA detection, then detection time is reduced to 60 hours and cost is lowered, but the method must overcome the complexity of multiple binding and amplification steps
Solution Approach 1:
The patent merges multiple functions into a single integrated detection system where CRISPR-Cas9 binding, rolling circle amplification, and fluorescent signal generation occur in a unified reaction protocol. This consolidation reduces the number of separate steps and reagents needed, simplifying the detection process while maintaining rapid turnaround time of 60 hours.
Solution Approach 2:
The patent introduces rolling circle amplification as an intermediary mechanism that bridges the CRISPR binding step and the fluorescent detection step. This intermediary amplification process efficiently converts the specific binding event into a detectable signal, simplifying the overall detection workflow while maintaining high specificity and rapid results.
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
The method significantly reduces detection time to 60 hours, lowers costs, and enhances specificity by targeting specific eccDNA sequences, achieving a detection limit of 1 fM.
Implementation Method 1
binding Cas9n-sgRNA to a target site of a target double-stranded circular DNA by using CRISPR-RCA technology
Implementation Method 2
binding the 40 nt single-stranded DNA probe to a non-complementary strand of sgRNA
Implementation Method 3
extending the non-complementary strand of sgRNA at the target site with Klenow large-fragment enzyme
Implementation Method 4
using Phi29 to perform rolling circle amplification on the single-stranded circular DNA obtained from the strand displacement
Implementation Method 5
binding Cas14a-sgRNA and a reporter probe to a target site of the rolling circle amplification product by using CRISPR-RCA technology
Implementation Method 6
by using Cas14a for its high-fidelity binding characteristics and the trans-cleavage activity after binding to the target site, the signal at the target site is amplified and the amplified signal is converted into a detectable fluorescence signal
Implementation Method 7
performing fluorescence detection under a fluorescence spectrophotometer
Data Source
AI summary
A rapid eccDNA detection method based on CRISPR-RCA technology is provided, which belongs to the field of biotechnology. The method includes the following steps: targeting eccDNA containing a target sequence by using a CRISPR-Cas9 system, carrying out single-stranded DNA probe-assisted enzyme modification at an incision of a target site, directly initiating rolling circle amplification on the target eccDNA at a constant temperature, then targeting and binding a single-stranded rolling circle amplification product by using a CRISPR-Cas14 system to activate the trans-cleavage activity of Cas14, thereby achieving secondary signal amplification.