CRISPR/Cas Chain Reaction System for High-Sensitivity Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR-based detection methods have low sensitivity at room temperature without target pre-amplification and lack a multiplexable, rapid, sensitive, inexpensive, and accessible testing solution for point-of-care diagnostics and environmental monitoring.
Innovation Solution
The CRISPR/Cas chain reaction (CCR) system amplifies detection sensitivity by using a primary CRISPR-based target detection system with secondary crRNAs, activators, and blocking moieties to create a cascade of activated Cas complexes that cleave probes, generating a detectable signal without the need for pre-amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-based detection is performed without target pre-amplification, then the detection process is simpler and faster, but the detection sensitivity is low
Solution Approach 1:
The patent applies preliminary action by designing blocking moieties that are pre-configured on secondary crRNAs or activators. These blocking moieties prevent premature binding, and are subsequently removed by activated Cas enzymes during the detection process. This pre-prepared blocking mechanism enables the system to achieve high sensitivity without requiring target pre-amplification, thus resolving the contradiction between detection speed and sensitivity.
2Measurement precision
If pre-amplification steps are added to improve detection sensitivity, then detection sensitivity increases, but the cost and complexity of the system increase
Solution Approach 1:
The patent implements self-service by designing a system where the activated Cas enzyme performs dual functions: it cleaves the target sequence and simultaneously removes the blocking moieties from secondary crRNAs or activators. This self-amplifying mechanism generates multiple activated complexes without requiring external pre-amplification steps, thereby achieving high detection sensitivity while maintaining system simplicity and reducing costs.
3Measurement precision
If pre-amplification steps are added to improve detection sensitivity, then detection sensitivity increases, but the time required for detection increases
Solution Approach 1:
The patent applies merging by combining the target cleavage function and the blocking moiety removal function into a single activated Cas enzyme complex. This unified mechanism allows the system to simultaneously achieve target detection and signal amplification in one step, eliminating the need for separate pre-amplification steps and reducing overall detection time while maintaining high sensitivity.
4Ease of operation
If conventional CRISPR detection systems are used, then the system is simple to operate, but the detection sensitivity at room temperature is low
Solution Approach 1:
The patent introduces an intermediary mechanism using blocking moieties that are temporarily attached to secondary crRNAs or activators. These blocking moieties act as mediators that prevent premature complex formation, and are subsequently removed by activated Cas enzymes to enable signal amplification. This intermediary approach maintains ease of operation while significantly improving detection sensitivity at room temperature.
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 CCR system significantly enhances detection sensitivity and specificity, allowing for the detection of attomolar levels of targets like SARS-CoV-2 and HIV within 60-90 minutes at room temperature, reducing the need for costly amplification steps and enabling point-of-care diagnostics.
Implementation Method 1
a primary crRNA/Cas complex, each primary crRNA having a primary guide sequence configured to bind a target, such that binding of the primary crRNA/Cas complex to the target activates the trans-cleavage activity
Implementation Method 2
each blocking moiety including a cleavable sequence configured to be cleaved by an activated Cas enzyme of an activated primary crRNA/Cas complex or an activated secondary crRNA/Cas complex, such that cleavage of the cleavable sequence of the blocking moiety releases the blocking moiety
Implementation Method 3
a plurality of secondary crRNAs capable of forming a complex with one of the Cas enzymes to form a secondary crRNA/Cas complex, each secondary crRNA comprising a secondary guide sequence configured to bind an activator
Implementation Method 4
a plurality of probes, each probe comprising an oligonucleotide element labeled with a detectable label, wherein the probe is configured to be cleaved by any of the activated Cas enzymes of the activated primary crRNA/Cas complexes or the activated secondary crRNA/Cas complexes to generate a detectable signal or a detectable molecule
Data Source
AI summary
The present disclosure provides CRISPR/Cas chain reaction (CCR) systems and methods for amplifying the detection sensitivity of a primary CRISPR-based target detection (CBTD) system for detecting targets. Also described are methods of using CCR systems to amplify the detection sensitivity of primary CBTD systems to detect a target without a target preamplification step. Multiplexed CBTD systems for detecting a target using two different Cas enzyme systems are provided.