One-Step Nucleic Acid Detection Using CRISPR-Cas12a Mediator
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Solution Overview
Problem
Current nucleic acid diagnostic tests for SARS-CoV-2 are limited by the need for skilled personnel, equipment, and long sample-to-answer times, with isothermal amplification methods like RPA and LAMP suffering from high false positive rates and complex operation, while CRISPR-based methods require optimized conditions to balance amplification and cleavage efficiently.
Innovation Solution
A one-step nucleic acid detection assay using a mixture of polymerase, deoxynucleoside triphosphates, primers, CRISPR-associated nuclease, and guide RNA, where suboptimal PAM sequences and modified nucleotides or guide RNAs are used to reduce Cas nuclease activity, allowing for efficient amplification and cleavage in a single reaction, thereby accelerating detection and improving sensitivity and reliability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If isothermal amplification assays (RPA/LAMP) are used for rapid nucleic acid detection, then the detection speed and accessibility are improved, but the false positive rate increases due to nonspecific amplification
Solution Approach 1:
The patent introduces CRISPR-Cas12a system as an intermediary verification layer between amplification and detection. The Cas12a-nuclease acts as a mediator that specifically recognizes and cleaves only those amplified products containing the target sequence, thereby eliminating false positives from nonspecific amplification while preserving the rapid isothermal amplification advantage
Solution Approach 2:
The patent extracts the specific recognition function from the amplification process by separating it into a distinct CRISPR-based detection step. The guide RNA is designed to specifically bind to the target sequence, extracting the specificity requirement from the general amplification reaction and placing it in a dedicated recognition step that eliminates false positives
2Reliability
If two-step CRISPR-based detection methods (SHERLOCK/DETECTR) are used to improve sensitivity and reduce false positives, then the detection reliability is improved, but the operation complexity and time consumption increase
Solution Approach 1:
The patent merges the amplification step and CRISPR detection step into a single one-pot reaction system. Both the isothermal amplification reagents (polymerase, primers, dNTPs) and CRISPR components (Cas12a, guide RNA, PAM sequence) are combined in one reaction mixture, eliminating the need for separate preparation and transfer steps between amplification and detection
Solution Approach 2:
The patent designs a universal one-pot reaction system that simultaneously performs multiple functions: isothermal amplification of target nucleic acid, CRISPR-mediated specific recognition, and collateral cleavage of reporter molecules. This multi-functional system replaces the specialized two-step workflow, reducing operational complexity while maintaining detection reliability
3Reliability
If CRISPR-Cas12a cleavage is enhanced to improve specificity, then the false positive rate is reduced, but the amplification efficiency decreases due to premature cleavage
Solution Approach 1:
The patent introduces a PAM (protospacer adjacent motif) sequence as a local quality marker that distinguishes target-containing amplicons from nonspecific products. The Cas12a system is programmed to recognize and cleave only at sites with the specific PAM sequence, allowing enhanced specificity through localized recognition rather than global amplification inhibition
Solution Approach 2:
The patent incorporates the PAM sequence into the primer design during the preliminary amplification setup. This preliminary incorporation ensures that only amplicons generated from specific target sequences contain the PAM motif, allowing Cas12a to selectively cleave these products later without interfering with the amplification process itself
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 assay achieves fast, sensitive, and reliable detection of nucleic acids within 15-20 minutes with comparable sensitivity to qPCR, offering a simple, instrument-free, and flexible alternative for point-of-care screening with reduced false positives and negatives.
Implementation Method 1
isothermal amplification assays such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)
Implementation Method 2
Cas12a, Cas12b and Cas13a have been repurposed as promising diagnostic tools owing to their collateral degradation of ssDNA or ssRNA
Data Source
AI summary
Compositions and methods are provided for simple, instrument-free and sensitive methods that enable rapid, point-of-care detection of nucleic acid molecules of interest. This is based on a surprising discovery that the relative efficiencies of amplification and CRISPR-based cleavage and detection can be tuned to favor amplification until sufficient amplified products are generated to enable detection. Example approaches include design of guide RNA and primers to target nonoptimal PAM sequences, or sequence-engineering Cas nucleases to reduce activities informing a ribonucleoprotein with the guide RNA or binding to or cleaving the substrate nucleic acid.