Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection

The ongoing arthropod-borne Chikungunya virus (CHIKV) highlights the requirements of rapid and accurate diagnostic methods to enhance the epidemic control. CRISPR diagnostic (CRISPR-Dx) technology holds promise, but the development of a highly efficient one-pot diagnostic system usually requires fine-tuning of the balance between isothermal amplification and Cas cleavage procedures. Here, we describe a simple method (psHOLMES) to create one-pot, two-step CRISPR-Dx systems, using photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity. Cas12a activity is first inactivated via binding of ppPS-DNA during the target sequence amplification procedure, which is then reactivated by ultraviolet (UV)-mediated photolysis of ppPS-DNA after amplification, triggering Cas12a trans-cleavage reactions. psHOLMES demonstrates attomolar sensitivity for CHIKV RNA detection and zero cross-reactivity against other related arboviruses. When applied to clinical samples, psHOLMES achieved 100% (50/50) accuracy and could detect CHIKV within 30 min. As traditional efforts for fine-tuning Cas cis-cleavage activity can be omitted, psHOLMES thus enables rapid development of one-pot CRISPR-Dx systems for clinical applications.