CRISPR Tick-Borne Disease Assays for Sensitive Strain Detection
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Solution Overview
Problem
Current diagnostic assays for tick-borne diseases, such as Lyme disease, babesiosis, and anaplasmosis, are insensitive, unreliable, and lack sequence- and strain-specific diagnostics, particularly in early infections and immunocompromised individuals, necessitating the need for rapid, sensitive, and multiplexed point-of-care diagnostics.
Innovation Solution
A nucleic acid detection system utilizing a CRISPR system with effector proteins and guide RNAs designed to bind specifically to tick-borne disease targets, combined with RNA-based masking constructs and nucleic acid amplification reagents, integrated into a lateral flow device for rapid detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current serological testing methods are used for Lyme disease detection, then the testing can be performed with standard equipment, but the sensitivity is poor especially in early infection and it cannot reliably distinguish between acute and prior infection or different strains
Solution Approach 1:
The patent uses CRISPR-Cas13a system as an intermediary between target pathogen RNA and detection signal. The Cas13a protein, when guided by crRNA to match target sequences, activates collateral RNase activity that cleaves reporter RNA molecules, producing a detectable fluorescent signal. This intermediary system enables highly sensitive and specific detection while maintaining operational simplicity through a single-tube isothermal amplification approach.
Solution Approach 2:
The patent employs isothermal amplification at constant temperature (20-40°C) rather than requiring thermal cycling. By changing the detection parameter from temperature-dependent PCR to isothermal RPA/REACT, the system achieves high sensitivity without complex thermal control equipment, making it suitable for point-of-care settings while maintaining the ability to distinguish acute from prior infections through sequence-specific detection.
2Loss of information
If sequence-specific diagnostics are developed to identify different strains and variants, then the understanding of pathogenesis improves, but the diagnostic assay complexity increases
Solution Approach 1:
The patent divides the detection task into multiple independent crRNA guides, each targeting specific strain-specific sequences. Different crRNAs can be included in the same reaction to detect multiple strains simultaneously. This segmentation approach allows strain identification without requiring complex multiplexing infrastructure, as each crRNA-guide complex operates independently to provide sequence-specific detection.
Solution Approach 2:
The CRISPR-Cas13a platform serves multiple functions: it amplifies target RNA isothermally, provides sequence-specific detection through guide RNA matching, and generates detectable signals through collateral RNase activity. This multi-functionality allows a single assay system to perform both pathogen detection and strain identification, reducing overall diagnostic complexity while maintaining high information content about the infecting organism.
3Loss of time
If rapid point-of-care diagnostics are implemented for tick-borne diseases, then treatment guidance can be provided immediately, but the detection sensitivity and sequence-specificity may be compromised
Solution Approach 1:
The patent performs preliminary isothermal amplification of target RNA in the same tube before CRISPR-based detection. By preparing amplified target material in advance within the reaction mixture, the system ensures sufficient target copy numbers for sensitive detection without requiring separate extraction and amplification steps. This preliminary action enables rapid point-of-care testing while maintaining high detection accuracy through sequence-specific CRISPR recognition.
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 system provides attomolar sensitivity for detecting tick-borne pathogens, differentiating targets based on single base pair differences, and is suitable for point-of-care applications, including freeze-dried formats for convenient distribution.
Implementation Method 1
a detection CRISPR system having an effector protein and one or more guide RNAs each designed to bind to corresponding target molecules that are diagnostic for a tick-borne disease state
Implementation Method 2
an RNA-based masking construct
Data Source
AI summary
Provided herein is a nucleic acid detection system comprising a detection CRISPR system having an effector protein and one or more guide RNAs each designed to bind to corresponding target molecules that are diagnostic for a tick-borne disease state; and an RNA-based masking construct. In some embodiments, the detection system of may comprise i) two or more CRISPR systems, each CRISPR system comprising an effector protein and a guide RNA designed to bind to a corresponding target molecule that is diagnostic for a tick-borne disease state; and ii) a set of detection constructs, each detection construct comprising a cutting motif sequence that is preferentially cut by one of the activated CRISPR effector proteins. Exemplary tick-borne detectable microbes include Babesia microti, Anaplasma phagocytophilum, and Borrelia miyamotoi.