CRISPR-Cas13a Diagnostic System for Attomolar Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in achieving high sensitivity and specificity while being cost-effective and portable, particularly in point-of-care settings, due to trade-offs between sensitivity, specificity, simplicity, and speed.
Innovation Solution
A CRISPR-based system comprising an effector protein and guide RNAs designed to bind target molecules, combined with an RNA-based masking construct and optional nucleic acid amplification reagents, for sensitive and specific detection of nucleic acids in a portable platform.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR approaches are used for nucleic acid detection, then sensitivity is improved, but device complexity and cost increase due to expensive instrumentation and complex operation requirements
Solution Approach 1:
The patent replaces complex mechanical/optical instrumentation (qPCR machines) with a biochemical system based on CRISPR-Cas13a effector protein that produces a detectable signal through RNA cleavage activity, eliminating the need for expensive thermal cyclers and fluorescence detection systems while maintaining high sensitivity
Solution Approach 2:
The CRISPR-Cas13a system performs self-detection through its inherent RNA-guided RNA cleavage activity, where the effector protein directly interacts with target RNA and produces a measurable signal without requiring external instrumentation or complex operational procedures
2Device complexity
If isothermal nucleic acid amplification with portable platforms is used, then device complexity is reduced for point-of-care use, but detection sensitivity decreases
Solution Approach 1:
The patent combines isothermal amplification (RPA or NASBA) with CRISPR-Cas13a detection in a single integrated system, where the amplification step generates sufficient target RNA that is then detected by the portable CRISPR assay, achieving both high sensitivity and portability by merging two complementary techniques
3Ease of operation
If conventional detection methods are used, then ease of operation is maintained, but detection specificity for single-base differences is insufficient
Solution Approach 1:
The patent introduces a local quality requirement by designing guide RNAs with specific sequences that must match the target RNA with perfect complementarity, particularly at critical positions near the cleavage site, thereby achieving single-base specificity through localized sequence requirements rather than overall complexity
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 enables attomolar sensitivity and differentiation of targets based on single-base pair differences, suitable for various healthcare applications, including viral detection and disease-associated cell-free DNA, with potential for point-of-care use.
Implementation Method 1
a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules
Implementation Method 2
a primer comprising an RNA polymerase promoter. In certain embodiments, sample nucleic acids are amplified to obtain a DNA template comprising an RNA polymerase promoter, whereby a target RNA molecule may be generated by transcription
Implementation Method 3
The nucleic acid may be RNA and amplified by a reverse transcription method as described herein
Data Source
AI summary
The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect broth DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.