CRISPR Nickase Isothermal Amplification for Sensitive Diagnostics
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Solution Overview
Problem
Current nucleic acid amplification methods for diagnostics face challenges in achieving high sensitivity and specificity while being cost-effective and suitable for point-of-care settings, often requiring complex instrumentation and having limited applications due to trade-offs in sensitivity and simplicity.
Innovation Solution
A method utilizing a CRISPR-based nickase system with dual CRISPR/Cas complexes and a polymerase for isothermal amplification, where the CRISPR/Cas complexes nick the target nucleic acid strands, allowing for repeated extension and amplification under constant temperature, using Cas9, Cpf1, or C2c1 nickases and specific guide molecules to target specific locations on the nucleic acid.
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 complex instrumentation requirements
Solution Approach 1:
The patent replaces complex mechanical thermal cycling instrumentation with a biochemical isothermal amplification system using CRISPR-Cas nickases and polymerases that function at constant temperature, eliminating the need for sophisticated temperature control machinery while maintaining high detection sensitivity
Solution Approach 2:
The patent introduces CRISPR-Cas nickase complexes as intermediary molecular machines that perform site-specific nicking of nucleic acids at constant temperature, serving as a mediator between the target nucleic acid and the amplification process without requiring complex thermal cycling equipment
2Device complexity
If isothermal nucleic acid amplification methods are used, then device complexity is reduced, but detection sensitivity is limited
Solution Approach 1:
The patent divides the amplification process into distinct functional modules: CRISPR-Cas nickase complexes for site-specific nicking, polymerases for extension, and guide RNAs for target recognition, allowing each component to be optimized for high sensitivity while maintaining isothermal operation and simple instrumentation
Solution Approach 2:
The patent creates composite molecular systems combining CRISPR-Cas proteins with polymerases and guide RNAs to form multifunctional nickase complexes that simultaneously provide target specificity, nicking activity, and amplification capability, achieving high detection sensitivity through composite molecular architecture
3Measurement precision
If multiple sets of primers and initial denaturation steps are required, then amplification specificity is improved, but ease of operation deteriorates due to complex procedures
Solution Approach 1:
The patent designs universal CRISPR-Cas nickase complexes with guide RNAs that can target multiple locations on the nucleic acid template simultaneously, eliminating the need for multiple primer sets and complex denaturation steps while maintaining high amplification specificity through programmable guide RNA sequences
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 enables sensitive and specific amplification of nucleic acids at low costs, suitable for portable platforms, achieving attomolar or femtomolar detection sensitivity and simplifying the process for point-of-care diagnostics.
Implementation Method 1
a first guide molecule that guides the first CRISPR/Cas complex to a first location on the target nucleic acid, and a second guide molecule that guides the second CRISPR/Cas complex to a second location of the target nucleic acid
Implementation Method 2
an amplification CRISPR system, the amplification CRISPR system comprising a first and second CRISPR/Cas complex, the first CRISPR/Cas complex comprising a first Cas-based nickase and a first guide molecule
Implementation Method 3
a polymerase; amplifying the target nucleic acid; further amplifying the target nucleic acid by repeated extension and nicking under isothermal conditions
Data Source
AI summary
The embodiments disclosed herein utilized RNA targeting effectors to provide robust CRISPR-based nucleic acid amplification methods and systems. Embodiments disclosed herein can amplify both double-stranded and single-stranded nucleic acid targets. Moreover, the embodiments disclosed herein can be combined with various detection platforms, for example, CRISPR-SHERLOCK, to achieve detection and diagnostic with attomolar sensitivity. 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.