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Room temperature CRISPR diagnostics for low-resource settings.

Eric A Nalefski¹, Selma Sinan², Jason L Cantera³

¹Global Health Labs, Inc, Bellevue, WA, 98007, USA. eric.nalefski@ghlabs.org.

Scientific Reports

|January 31, 2025

Summary

This summary is machine-generated.

This study introduces a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR diagnostic assay. This innovation simplifies diagnostics, making them more accessible for low-resource settings by eliminating heating requirements.

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Area of Science:

Molecular Biology
Biotechnology
Diagnostics

Background:

Elevated temperatures and complex sample preparation increase diagnostic costs and hinder use in low-resource settings.
Current recombinase polymerase amplification (RPA) and Cas12a-based diagnostics often require temperatures above 37°C, limiting their field applicability.

Purpose of the Study:

To develop a simplified, one-pot, room temperature diagnostic assay using recombinase polymerase amplification (RPA) and CRISPR technology.
To identify and utilize a Cas12a enzyme variant effective at 25°C for improved diagnostic performance in resource-limited environments.

Main Methods:

Investigated RPA amplification efficiency at 25°C versus 37°C.
Assessed the performance of various Cas12a orthologs at reduced temperatures.
Identified TsCas12a as a highly active nuclease at 25°C.
Developed and optimized a single-protocol RPA-TsCas12a reaction for room temperature use.
Performed quantitative kinetic analysis of nuclease activation and trans-cleavage activity.

Main Results:

RPA amplification significantly decreased at 25°C compared to 37°C.
Most tested Cas12a orthologs showed compromised activity below 37°C.
TsCas12a demonstrated high nuclease activity at 25°C.
The developed RPA-TsCas12a reaction effectively detected HPV-16 in crude cervical swab samples at room temperature.
High sensitivity and specificity were achieved with both optical and lateral flow readouts.

Conclusions:

A one-pot, room temperature RPA-TsCas12a assay overcomes limitations of existing diagnostics, reducing complexity and equipment needs.
This approach offers a promising solution for affordable and accessible diagnostics in low- and middle-income countries.
Fast nuclease activation is crucial for effective room temperature diagnostic applications.