Leveraging high temperature for enhanced and faster detection of SARS-CoV-2 and its Omicron variant with CRISPR-AsCas12a.

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.