Cas13-crRNA SARS-CoV-2 RNA Detection for Rapid Field Screening
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Solution Overview
Problem
Current methods for detecting SARS-CoV-2 RNA are slow and require laboratory access, failing to provide timely results at crucial community convergence points, and are ineffective in identifying asymptomatic carriers or variants, leading to uncontrolled spread of the virus.
Innovation Solution
A method using Cas13 protein and CRISPR guide RNA (crRNA) to detect and quantify SARS-CoV-2 RNA in samples, including saliva, sputum, and nasopharyngeal materials, without the need for reverse transcription, and capable of distinguishing variants and mutants, utilizing a reporter RNA that generates a detectable signal upon cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR method is used for SARS-CoV-2 detection, then detection accuracy is improved, but turn-around time increases and field deployability deteriorates
Solution Approach 1:
The invention extracts and utilizes the collateral RNA cleavage activity of Cas13 protein separately from its target-specific binding function. The crRNA-Cas13 complex specifically binds to SARS-CoV-2 RNA, while the cleavage of reporter RNA is a separate detectable event, enabling rapid detection without lengthy amplification cycles
Solution Approach 2:
The invention introduces reporter RNA as an intermediary molecule that translates the specific Cas13-SARS-CoV-2 RNA interaction into a detectable signal. The reporter RNA is cleaved by activated Cas13, producing a measurable output that indicates SARS-CoV-2 presence without requiring direct detection of the viral RNA itself
2Measurement precision
If PCR method is used for SARS-CoV-2 detection, then detection accuracy is improved, but device complexity and laboratory equipment requirements increase
Solution Approach 1:
The Cas13 protein system is self-activating upon binding to SARS-CoV-2 RNA via crRNA. The collateral cleavage activity inherently produces detectable signals without requiring external amplification machinery or complex laboratory equipment, enabling the system to serve itself and perform detection in simplified settings
Solution Approach 2:
The invention replaces the mechanical/thermal cycling system of PCR with a biochemical recognition system. Instead of requiring thermal cycled amplification and sophisticated instrumentation, the Cas13-crRNA complex performs specific RNA recognition and activation at constant temperature, substituting complex mechanical systems with simpler biochemical reactions
3Ease of operation
If traditional screening methods are used, then ease of operation is improved, but detection reliability deteriorates due to inability to identify asymptomatic carriers
Solution Approach 1:
The invention employs disposable crRNA-Cas13 complexes that are pre-assembled and stable at room temperature. These single-use detection reagents eliminate the need for expensive, complex laboratory equipment while maintaining high detection reliability, making them suitable for widespread deployment in various settings including field operations
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
Enables rapid, field-deployable detection of SARS-CoV-2 RNA with high sensitivity and specificity, allowing for timely identification of infected individuals and variants, even at low viral loads, without the need for laboratory equipment.
Implementation Method 1
incubating a sample suspected of containing SARS-CoV-2 RNA with a Cas13 protein, at least one CRISPR guide RNA (crRNA), and a reporter RNA
Implementation Method 2
a reporter RNA that generates a detectable signal upon cleavage
Data Source
AI summary
The present disclosure relates to methods using CRISPR-Cas13 enzyme, complexed with SARS-CoV-2 crRNA guide RNAs to detect and quantify the presence of SARS-CoV-2 RNA in a sample with enhanced specificity and sensitivity. These methods can be used to diagnose SARS-CoV-2 infection, quantify the concentration of SARS-CoV-2 RNA present in a sample, identify the presence of different SARS-CoV-2 splice variants, subtypes, or mutations, and to monitor reactivation of SARS-CoV-2 transcription.