Proximity-Activated CRISPR Assays for Sensitive Analyte Detection
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Solution Overview
Problem
Existing diagnostic methods for detecting molecular analytes such as antibodies, proteins, and small molecules are limited by low sensitivity, specificity, and require multiple processing steps, making them unsuitable for low-cost, point-of-care formats.
Innovation Solution
A method using CRISPR-Cas systems with collateral ssDNase or ssRNase activity, combined with synthetic nucleotide sequences and guide RNAs, to detect molecular analytes through proximity-driven activation and cleavage of reporter constructs, enabling rapid and sensitive detection without thermal cycling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard immunoassays are used to detect molecular analytes, then detection capability is maintained, but sensitivity and specificity are reduced
Solution Approach 1:
The patent employs CRISPR-Cas nucleases as intermediary molecules that bridge the detection of molecular analytes (antibodies, proteins, small molecules) with signal generation. The Cas nuclease complex mediates between the target analyte and the reporter construct, providing highly sensitive and specific detection through collateral ssDNase activity that amplifies the detection signal.
Solution Approach 2:
The patent changes the detection parameter from direct binding visualization to nucleic acid cleavage detection. By using CRISPR-Cas systems, the detection mechanism transitions from measuring molecular binding events to measuring enzymatic cleavage of DNA reporters, enabling attomolar detection limits and superior sensitivity/specificity.
2Measurement precision
If nucleic acid amplification techniques are combined with protein detection, then sensitivity is improved, but device complexity and processing steps increase
Solution Approach 1:
The patent merges protein detection with nucleic acid amplification into a single integrated assay. The CRISPR-Cas system combines the target recognition function (via guide RNA binding to analyte-specific sequences) with the amplification function (via collateral ssDNase activity) in one reaction, eliminating the need for separate detection and amplification steps.
Solution Approach 2:
The CRISPR-Cas nuclease system serves multiple functions simultaneously: it acts as a target recognizer through guide RNA, an amplification engine through collateral cleavage activity, and a signal generator through reporter construct degradation. This multi-functionality reduces overall assay complexity while maintaining high sensitivity.
3Measurement precision
If thermal cycling and multiple processing steps are used, then detection accuracy is improved, but ease of operation and cost are reduced
Solution Approach 1:
The patent replaces thermal cycling (a mechanical/thermal process) with isothermal enzymatic reactions. The CRISPR-Cas system performs target recognition and signal amplification at constant temperature through enzymatic mechanisms, eliminating the need for temperature variations and complex thermal cycling equipment, thereby simplifying operation and reducing costs.
4Measurement precision
If CRISPR-Cas systems are used for detection, then sensitivity reaches attomolar levels, but device complexity increases
Solution Approach 1:
The patent segments the detection system into distinct functional modules: guide RNA (for target recognition), CRISPR-Cas nuclease (for signal amplification), and reporter construct (for signal output). This modular segmentation allows each component to be optimized independently and simplifies the overall system design while achieving attomolar detection sensitivity.
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
Provides highly sensitive and specific detection of diverse molecular analytes, suitable for point-of-care diagnostics, without requiring complex instrumentation, and allowing multiplexed reactions for simultaneous detection of multiple analytes.
Implementation Method 1
a Cas nuclease that exhibits collateral ssDNase or ssRNase activity
Implementation Method 2
the gRNA forms a complex with the dsPAM sequence and the dsDNA spacer sequence
Implementation Method 3
binding of the first and second antigens to a target molecule
Implementation Method 4
The first ssDNA and the second ssDNA PAM sequences comprise complementary sticky ends and form a double-stranded PAM (dsPAM) sequence
Data Source
AI summary
Provided herein are methods and compositions for rapid, highly sensitive detection of molecular analytes such as antibodies, proteins, and small molecules using protein-driven nucleic acid assemblies to activate CRISPR-Cas nucleases. Also provided herein are uses of the sensitive analyte detection methods in an analyte detection platform and in convenient low-cost diagnostic assays such as lateral flow devices for point-of-care use.