CRISPR-Cas Transposase Biosensor for Nucleic Acid Detection
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Solution Overview
Problem
Current field-deployable nucleic acid diagnostic technologies for disease biosurveillance are limited by sensitivity, require target pre-amplification, involve complex steps like denaturation and manual handling, are not suitable for both RNA and DNA targets, and lack rapid reconfigurability.
Innovation Solution
The use of a point-of-need disposable 'FET Strip' (enzymatic) and an instrument-operated 'FET Multiplexor' (electronic) system, incorporating Cas 12, Cas 13, and RNA-guided transposase for nucleic acid detection, with a colorimetric lateral flow assay and ultrasensitive single-molecule field-effect transistors, along with a reporter screen and deep learning-based framework for guide RNA design.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If lateral flow assay (LFA) tests are used for simple field deployment, then device complexity is reduced, but sensitivity deteriorates and requires target pre-amplification or additional complex steps
Solution Approach 1:
The patent combines LFA simplicity with CRISPR-Cas diagnostic sensitivity by integrating Cas enzyme-based detection into the lateral flow assay format. This merging allows the device to maintain the ease of use and low complexity of LFA while achieving the high sensitivity typically associated with complex molecular diagnostic methods, eliminating the need for separate pre-amplification steps.
Solution Approach 2:
The diagnostic system is designed to detect multiple pathogen targets simultaneously using a single LFA device configuration. By incorporating multiplexed probe designs and using Cas enzymes that can target different nucleic acid sequences, the system achieves universal detection capability across various pathogens without requiring separate devices or complex sample processing for each target.
2Measurement precision
If Cas-mediated cutting is used for sensitive detection, then measurement precision is improved, but device complexity increases due to required pre-amplification and downstream amplification steps
Solution Approach 1:
The patent extracts and eliminates the need for separate pre-amplification and downstream amplification steps from the diagnostic workflow. By designing the CRISPR-Cas system to directly detect and cut target nucleic acids without requiring prior amplification, the method simplifies the overall process while maintaining sensitivity. The Cas enzyme-mediated cleavage itself serves as the detection mechanism, removing unnecessary intermediate steps.
3Measurement precision
If denaturation with heating equipment and manual steps are used, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The diagnostic system is designed to perform detection without requiring external heating equipment or extensive manual intervention. The CRISPR-Cas enzymes function at ambient or physiological temperatures, eliminating the need for thermal cycler or heating blocks. The system uses self-assembling components and automated fluid handling where possible, allowing the detection process to proceed with minimal user input and no specialized equipment beyond the LFA device itself.
4Adaptability or versatility
If Cas 12 and Cas 13 systems are used for detection, then adaptability is improved for detecting both RNA and DNA targets, but device complexity increases
Solution Approach 1:
The patent employs a universal LFA device platform that can detect both RNA and DNA targets by incorporating appropriate Cas enzymes (Cas 12 for DNA, Cas 13 for RNA) into the same diagnostic system. The device architecture, probe design, and fluid handling are standardized across different target types, allowing laboratories to deploy a single platform for multiple pathogen detection applications without requiring separate devices for RNA and DNA diagnostics.
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 enhances sensitivity and speed in gene-editing diagnostics, enabling direct detection of genomic targets without denaturation, and allows for rapid and sensitive identification of both DNA and RNA targets without the need for pre-amplification or complex manual steps.
Implementation Method 1
a guide RNA that comprises a nucleic acid sequence complementary to a gRNA target nucleic acid sequence
Implementation Method 2
a CRISPR RNA-guided Cas-transposase system that cuts and pastes donor sequences into dsDNA targets
Implementation Method 3
an array of single-molecule field-effect transistors (smFET) that are ultrasensitive, capable of detecting single-molecule binding events
Implementation Method 4
a colorimetric lateral flow assay (LFA) with multiple (e.g., 10) zones for target capture and detection
Data Source
AI summary
The present disclosure provides devices, systems, and methods for detection of nucleic acids based on CRISPR-Cas editing systems, for example for use in biosurveillance. Disclosed herein are systems and methods utilizing two devices: 1) a point of—need disposable “FET Strip” (enzymatic), and 2) an instrument-operated “FET Multiplexor” (electronic), to provide detection of a nucleic acid for biosurveillance.