CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a

Jeong Moon, Jiongyu Zhang, Xin Guan, Rui Yang, Chong Guo, Kurt T. Schalper, Lori Avery, David Banach, Rocco LaSala, Ranjit Warrier and Changchun Liu ()
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Jeong Moon: University of Connecticut Health Center
Jiongyu Zhang: University of Connecticut Health Center
Xin Guan: University of Connecticut Health Center
Rui Yang: University of Connecticut Health Center
Chong Guo: University of Connecticut Health Center
Kurt T. Schalper: University of Connecticut Health Center
Lori Avery: University of Connecticut Health Center
David Banach: University of Connecticut Health Center
Rocco LaSala: University of Connecticut Health Center
Ranjit Warrier: Centre for Infectious Disease Research in Zambia
Changchun Liu: University of Connecticut Health Center

Nature Communications, 2025, vol. 16, issue 1, 1-12

Abstract: Abstract The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.

Date: 2025
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DOI: 10.1038/s41467-025-64205-4

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