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CRISPR-Responsive Reprogrammable Label-Free Fluorescent Nanoclusters for ML-Assisted Pathogenic Genome Detection on

Emmett Hanson¹, Nabeel Kalla¹, Ram Jeevan Tharu¹

¹Department of Chemistry, University at Albany, State University of New York, 1400 Washington Avenue, Albany, NY, 12222, USA.

Small (Weinheim an Der Bergstrasse, Germany)

|March 4, 2025

Summary

This study presents a paper-based genome detection assay using DNA-templated silver nanoclusters and CRISPR-Cas12a technology. The assay accurately detects Salmonella genomes with high sensitivity and specificity, even in complex samples.

Keywords:

CRISPR fluorescence genomes sensors silver nanoclusters

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Area of Science:

Biotechnology
Nanotechnology
Genomics

Background:

Developing sensitive and specific methods for genome detection is crucial for diagnostics.
Existing methods may lack portability or require complex laboratory setups.

Purpose of the Study:

To develop a paper-based genome detection assay utilizing DNA-templated silver nanoclusters.
To leverage CRISPR-Cas12a for programmable and specific genome recognition.
To demonstrate the assay's capability for detecting Salmonella genomes.

Main Methods:

Utilized DNA-templated silver nanoclusters (DFN-1) with intrinsic fluorescence.
Employed CRISPR-Cas12a for targeted DNA cleavage, modulating nanocluster fluorescence.
Integrated isothermal amplification for enhanced sensitivity.
Applied machine learning algorithms for data analysis.

Main Results:

Successfully detected Salmonella genome in liquid and on filter paper discs.
Demonstrated programmable detection of two Salmonella serotypes by altering crRNA.
Achieved detection of as low as 33 copies of Salmonella genome.
Attained 100% prediction accuracy in analyzing 60 unknown samples using machine learning.

Conclusions:

The developed paper-based assay offers a sensitive, specific, and programmable platform for genome detection.
CRISPR-Cas12a integration enables precise targeting and fluorescence-based readout.
The assay shows potential for rapid and portable pathogen detection applications.