CRISPR/Cas12a Pathogen Detection System
Solution Overview
Problem
Current methods for detecting citrus greening and phytoplasma diseases are inadequate for early and reliable detection due to low pathogen titers in infected plants, limiting their effectiveness in preventing disease transmission and minimizing crop losses.
Innovation Solution
The use of a CRISPR/Cas12-based system for detecting citrus greening and phytoplasma pathogens, which involves contacting a sample with a type V CRISPR/Cas effector protein, a guide RNA, and a single-stranded DNA reporter oligonucleotide, enabling ultrasensitive detection at the attomolar level and facilitating high-throughput, field-deployable diagnostics.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection methods (electron microscopy, serology, DNA probes, ELISA, qPCR) are used, then the detection can be performed with available technology, but the detection sensitivity is insufficient for early infection stage due to low pathogen titer
Solution Approach 1:
The patent changes the detection parameter from conventional qPCR sensitivity to attomolar-level sensitivity by implementing a CRISPR/Cas12a-based detection system. The system uses a guide RNA to specifically bind to target DNA sequences of citrus greening pathogens, activating Cas12a to cleave fluorescent reporter molecules, producing a detectable signal at attomolar concentrations (10^-18 M), which is 100-1000 times more sensitive than conventional qPCR methods.
Solution Approach 2:
The patent introduces an intermediary CRISPR/Cas12a detection system between the pathogen DNA and the detection signal. The guide RNA acts as a mediator that specifically recognizes and binds to target DNA sequences, while Cas12a serves as an intermediary enzyme that amplifies the detection signal through collateral cleavage of fluorescent reporters, enabling ultrasensitive detection of low pathogen titers at early infection stages.
2Measurement precision
If high sensitivity detection is achieved, then early detection capability is improved, but the device complexity and field deployability may be compromised
Solution Approach 1:
The patent extracts the core detection function from complex laboratory infrastructure by implementing a standalone CRISPR/Cas12a detection system that can be deployed in the field. The system uses only essential components: guide RNA, Cas12a protein, target DNA, and fluorescent reporters, eliminating the need for complex qPCR instrumentation while achieving attomolar-level sensitivity.
Solution Approach 2:
The patent employs disposable, chemically synthesized guide RNAs and fluorescent reporter molecules that can be prepared in advance and used in field conditions without requiring expensive, maintainable equipment. The detection reagents are designed to be stable, easy to store, and suitable for one-time use in field-deployable diagnostics, reducing device complexity while maintaining high sensitivity.
3Productivity
If conventional detection methods are used, then the method is simpler to implement, but the throughput and field deployability are limited
Solution Approach 1:
The patent replaces mechanical and instrumentation-dependent detection methods (electron microscopy, ELISA, qPCR) with a chemistry-based CRISPR/Cas12a system that produces optical signals through fluorescent reporter cleavage. This substitution enables high-throughput screening using standard plate readers or lateral flow strips, improving both productivity and field deployability while maintaining ease of operation.
Solution Approach 2:
The patent designs a universal CRISPR/Cas12a detection platform that can detect multiple citrus greening pathogens and phytoplasmas by simply changing the guide RNA sequence. The system is compatible with various detection formats including fluorescence microplate readers and lateral flow immunostrips, providing multi-functional capability that enhances both high-throughput screening and field deployability without compromising ease of operation.
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 early and accurate detection of citrus greening and phytoplasma pathogens, significantly improving the ability to manage disease transmission and minimize crop losses by enhancing diagnostic sensitivity and field applicability.
Implementation Method 1
a guide RNA (comprising a region that binds to the type V CRISPR/Cas effector protein, and a spacer sequence that hybridizes with the target DNA)
Implementation Method 2
detecting a signal produced by cleavage (by the type V CRISPR/Cas effector protein) of the single stranded DNA reporter oligonucleotide
Implementation Method 3
the single stranded DNA reporter oligonucleotide includes a fluorescence-emitting dye pair (e.g., a fluorescence-emitting dye pair is a fluorescence resonance energy transfer (FRET) pair, a quencher/fluor pair)
Data Source
AI summary
The embodiments disclosed herein utilize type V CRISPR/Cas effector proteins to provide robust diagnostic assays with attomolar sensitivity. Embodiments disclosed herein can detect DNA targets from bacterial plant pathogens and can differentiate targets from non-targets based on single base pair differences. Such embodiments are useful in multiple scenarios involving bacterial plant pathogens such as Candidatus Liberibacter asiaticus and Candidatus Phytoplasma trifolii responsible for citrus greening and potato purple top, respectively.