Journal of pharmaceutical and biomedical analysisOct 23, 2026

Using higher temperatures to improve and speed up CRISPR detection of SARS-CoV-2 and its Omicron variant

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Oct 24, 2026

Abstract

Heating the wild-type AsCas12a protein to 65°C improves nucleic acid detection by 33-fold without causing false positives.

CRISPR-based methods show effectiveness in detecting SARS-CoV-2, comparable to gold-standard PCR.
Adjusting the temperature of Cas proteins enhances DNA recognition, improving diagnostic specificity and sensitivity.
Elevating the reaction temperature allows for faster nucleic acid detection within a narrow operational range.
A two-step method utilizing fluorescence and lateral flow strips can detect SARS-CoV-2 in as little as 5 minutes.
This approach successfully differentiates the Omicron variant from the wild-type strain using plasmid DNA.

Simplified

CRISPR-based methods are effective for detecting SARS-CoV-2, with performance comparable to gold-standard PCR. Developing simpler, faster, and more sensitive CRISPR-based diagnostics will accelerate their adoption in field. Adjusting the temperature at which Cas proteins operate is crucial for enhancing DNA recognition kinetics, thereby improving specificity, sensitivity, and practicality. However, optimization is challenging due to the narrow temperature range for activity of wild-type Cas orthologs. Here, we show that heating the wild-type AsCas12a protein above its optimal temperature, up to 65ºC, accelerates nucleic acid detection, reducing the limit of detection (LoD) by 33-fold without causing false positives. This effect was further enhanced by buffer optimization. We applied these conditions to develop a two-step fluorescence- and lateral flow strip-based method for detecting SARS-CoV-2 in as little as 5 min from LAMP amplicons, and demonstrated its applicability for discriminating the Omicron variant from the wild-type strain using plasmid DNA. Overall, these findings demonstrate that increasing reaction temperature is a simple, effective strategy to enhance CRISPR-Cas12a diagnostics for pathogens, without the need for protein engineering or thermophilic Cas12a orthologs.

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Using higher temperatures to improve and speed up CRISPR detection of SARS-CoV-2 and its Omicron variant

Abstract

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