CRISPR-Cas Biosensor for Antibiotic Detection

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Solution Overview

Problem

Current methods for detecting small molecules like antibiotics in water sources are limited by high detection limits, reliance on expensive instruments, biosafety concerns, and the need for complex equipment, making them impractical for field deployment and sensitive detection.

Innovation Solution

A CRISPR/Cas-based biosensor system using synthetic double-stranded DNA substrates with specific operator sequences and Cas nuclease exhibiting collateral single-stranded nucleic acid cleavage activity, which detects target molecules by cleaving a reporter construct, allowing for sensitive and modular detection on both fluorescence plate readers and lateral flow assays.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If CRISPR/Cas-based biosensor system is used, then detection sensitivity is improved (detection limits as low as 0.9-4 nM), but device complexity increases due to multiple components (Cas nuclease, gRNA, dsDNA substrate, ssDNA reporter)

Engineering Contradiction:

Improvedetection sensitivityVSAvoidsystem complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The detection system is divided into distinct functional modules: Cas nuclease enzyme, guide RNA (gRNA), double-stranded DNA substrate with operator sequence, and single-stranded DNA reporter construct. Each component performs a specific function and can be independently optimized or replaced, managing complexity through functional segmentation while maintaining high detection sensitivity

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The CRISPR/Cas system serves multiple functions: specific target recognition through gRNA-DNA hybridization, enzymatic cleavage activation upon target binding, and collateral cleavage of reporter DNA for signal generation. This multi-functionality in a single system achieves high sensitivity without requiring multiple separate detection systems

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Ease of operation

If field-deployable detection is implemented, then ease of operation is improved (portable, no expensive instruments), but measurement precision may worsen (field conditions vs controlled lab environment)

Engineering Contradiction:

Improvefield deployabilityVSAvoiddetection accuracy

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The system uses disposable, non-instrumented readout methods such as lateral flow strips that provide visible results without requiring expensive or sophisticated equipment. The assay components can be prepared as single-use kits, enabling field deployment while maintaining reliable detection through simple visual interpretation of results

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

Solution Approach 2:

The system incorporates controls and optimization of reaction conditions (temperature, pH, buffer composition) that allow the assay to function reliably across varying field conditions. The CRISPR/Cas reaction parameters are tuned to maintain precision despite environmental variations, bridging the gap between controlled lab settings and field deployment

Inventive Principle:

Principle #35Parameter changes

3Productivity

If collateral cleavage mechanism is used, then productivity is improved (signal amplification), but loss of substance increases (consumption of reporter DNA)

Engineering Contradiction:

Improvesignal amplificationVSAvoidreporter DNA consumption

Core Design Contradiction:

ProductivityVSLoss of substance

Solution Approach 1:

The system separates the target recognition function (Cas nuclease-gRNA complex binding to dsDNA substrate) from the signal generation function (collateral cleavage of ssDNA reporter). This extraction allows the cleavage activity to be directed at an abundant reporter molecule rather than the limited target, amplifying the signal while minimizing consumption of the actual target analyte

Inventive Principle:

Principle #2Taking out (Extraction)

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 achieves detection limits as low as 0.9-4 nM for various small molecules, including antibiotics, and can function in different water samples, providing a cost-effective and field-deployable solution for monitoring small molecule contaminants.

Implementation Method 1

a Cas nuclease that exhibits collateral single-stranded DNase (ssDNase) activity

Methodology Applied

Scientific EffectCollateral cleavage:

Implementation Method 2

Cas nuclease that exhibits collateral single-stranded DNase (ssDNase) activity

Methodology Applied

Scientific EffectNuclease activity: Enzyme

Implementation Method 3

a guide RNA (gRNA), wherein the gRNA hybridizes the reverse complement of the operator sequence or the operator sequence

Methodology Applied

Scientific EffectHybridization:

Implementation Method 4

a synthetic double-stranded DNA (dsDNA) substrate comprising an operator sequence specific to a binding protein

Methodology Applied

Scientific EffectProtein-DNA binding:

Data Source

PatentUS20240094229A1Crispr-cas /transcription factor-based competition assay for detection of molecular analytes

Publication Date: 2024.03.21 THE ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIV OF ARIZONA

AI summary

Provided herein are highly modular and sensitive biosensors for small molecule detection using CRISPR/C as enzymes having collateral single-stranded nucleic acid cleavage activity. Also provided herein are uses of the biosensors in detection platforms for convenient, low-cost assessment of safety and purity of consumable samples, environmental samples, and agricultural products.