CRISPR/Cas Nucleic Acid Quantitation Without Amplification
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Solution Overview
Problem
Current methods for quantitating nucleic acids require amplification prior to detection, which can be cumbersome and may introduce biases, and there is a need for assays that can quantify nucleic acids without amplification.
Innovation Solution
A method using CRISPR/Cas effector proteins, guide RNAs, and reporter nucleic acids in nanovolumes to directly quantify nucleic acids by measuring cleavage signals without amplification, allowing for precise quantitation of target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid amplification is performed prior to detection, then detection sensitivity is improved, but assay complexity and time are increased
Solution Approach 1:
The patent extracts and eliminates the amplification step from the traditional nucleic acid detection workflow. By using CRISPR/Cas effector proteins that can directly detect and cleave target nucleic acids without prior amplification, the method removes this complex intermediate step while maintaining detection capability through direct binding and signal generation.
Solution Approach 2:
The patent replaces the mechanical/chemical amplification process (PCR) with a biological recognition system (CRISPR/Cas effector-protein guide RNA complex). This substitution allows direct detection of nucleic acids through specific binding and cleavage events, eliminating the need for thermal cycling and enzymatic amplification.
2Measurement precision
If nucleic acid amplification is performed prior to detection, then detection sensitivity is improved, but assay time is increased
Solution Approach 1:
The patent removes the time-consuming amplification step by using CRISPR/Cas effector proteins that can directly bind to and cleave target nucleic acids. This extraction of the amplification step eliminates the thermal cycling time and intermediate processing steps, allowing for rapid direct detection.
Solution Approach 2:
The patent skips the amplification intermediate step and goes directly from sample preparation to detection. The CRISPR/Cas effector protein complex rapidly binds to the target nucleic acid and generates a detectable signal through cleavage, rushing through the detection process without the time required for amplification.
3Measurement precision
If nucleic acid amplification is performed prior to detection, then detection capability is improved, but bias in quantitation is introduced
Solution Approach 1:
The patent extracts the amplification step that introduces bias and replaces it with direct detection using CRISPR/Cas effector proteins. By detecting nucleic acids in their native state without amplification, the method eliminates the biases introduced by amplification efficiency variations, primer specificity, and platform-dependent effects.
Solution Approach 2:
The patent uses guide RNA molecules that are copied or replicated during the detection process to ensure consistent and unbiased detection. The guide RNA binds to the target nucleic acid and facilitates cleavage by the CRISPR/Cas effector protein, providing a reliable copying mechanism that maintains quantitation accuracy without amplification biases.
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
Enables rapid and accurate quantitation of nucleic acids, such as viral nucleic acids, in samples within 30-90 minutes without the need for amplification, providing a reliable basis for diagnostics and epidemiological predictions.
Implementation Method 1
a guide sequence that is capable of hybridizing with the target nucleic acid
Implementation Method 2
measuring detectable signals detected from the at least two nanovolumes and generated by cleavage of the reporter nucleic acid by the CRISPR/Cas effector protein
Data Source
AI summary
Provided herein are compositions, systems, and methods comprising effector proteins (e.g., CRISPR-associated (Cas) proteins), and uses thereof. Various compositions, systems, and methods of the present disclosure may leverage the activities of these effector proteins for the detection and quantitation of nucleic acids.