CRISPR Cas RNP Signal Amplification for Direct Nucleic Acid Detection
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Solution Overview
Problem
Current CRISPR-Cas diagnostic systems are limited by sensitivity levels that are outside the range required for diagnostic purposes, and they often rely on pre-amplification of target sequences, which complicates direct point-of-care applications.
Innovation Solution
A method and kit for detecting nucleic acids using a Cas-signal amplification strategy that involves adding Cas ribonucleoprotein (RNP) with collateral activity, an amplifier, a reporter substrate, and optionally an exonuclease or polymerase to a sample, without the need for amplifying the nucleic acid of interest.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If pre-amplification of target sequences is performed to improve sensitivity, then detection sensitivity is improved, but device complexity and operational complexity increase
Solution Approach 1:
The patent introduces an intermediary amplification mechanism where the first Cas RNP acts as a mediator to activate the second Cas RNP, which then amplifies the signal. This intermediary approach allows sensitivity enhancement without requiring complex external amplification equipment, as the biological molecules themselves perform the amplification function.
Solution Approach 2:
The system employs self-service amplification where the Cas RNP complexes themselves perform the signal amplification without external intervention. The first Cas RNP activates the second Cas RNP, which then autonomously amplifies the signal through its collateral activity, eliminating the need for separate amplification devices or procedures.
2Measurement precision
If pre-amplification steps are added to improve sensitivity, then detection sensitivity is improved, but the procedure time and operational simplicity deteriorate
Solution Approach 1:
The patent merges the detection and amplification functions into a single integrated reaction system. The first and second Cas RNPs operate simultaneously in the same reaction mixture, allowing signal amplification to occur during the detection phase itself rather than requiring separate pre-amplification steps, thereby reducing total procedure time.
Solution Approach 2:
The system performs preliminary activation where the first Cas RNP pre-activates the second Cas RNP before the main detection reaction begins. This preliminary action ensures that the amplification mechanism is already in place and ready to operate immediately upon target detection, eliminating delays associated with sequential processing.
3Ease of operation
If standard CRISPR-Cas systems are used without amplification, then operational simplicity is maintained, but detection sensitivity is insufficient for diagnostic purposes
Solution Approach 1:
The patent introduces an intermediary amplification mechanism where the first Cas RNP acts as a mediator to activate the second Cas RNP, which then amplifies the signal. This intermediary approach allows sensitivity enhancement without requiring complex external amplification equipment, as the biological molecules themselves perform the amplification function.
Solution Approach 2:
The system employs self-service amplification where the Cas RNP complexes themselves perform the signal amplification without external intervention. The first Cas RNP activates the second Cas RNP, which then autonomously amplifies the signal through its collateral activity, eliminating the need for separate amplification devices or procedures.
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 approach allows for the detection and quantification of nucleic acids in very small amounts, achieving an authentic exponential chain reaction with improved sensitivity and reduced background signals, enabling simple, fast, and on-site detection.
Implementation Method 1
a robust collateral nuclease activity that some of these Cas proteins acquire when they cleave their target sequence. This collateral activity consists in a sequence-independent unscheduled endonucleolytic degradation, normally of single-stranded nucleic acids of the same type as the sequence-specific target.
Implementation Method 2
the specificity and versatility conferred by their mechanism of target recognition and cleavage, which is directed by base-pair hybridization with a guide RNA (gRNA) sequence
Data Source
AI summary
A method for detecting the presence of a nucleic acid sequence in a sample using CRISPR technology. The method allows the detection of nucleic acid amounts as low as 100 fM with a high signal-to-noise ratio and without the need to perform any amplification of the nucleic acid of interest. A kit to carry out the method for detecting the presence of a nucleic acid sequence in a sample, and to the use of the method or the kit of the invention for the diagnosis or prognosis of a disease or condition.