CRATS: a one-pot CRISPR-Cas12b and RPA combined assay with a temperature switch for highly sensitive detection of monkeypox virus

Abstract

CRISPR-Cas nucleases have been extensively used in molecular detections, especially highly sensitive nucleic acid detections. In these detections, Cas nucleases are programmed by the guide RNA to respond to the detection targets and cleave the chemically labelled molecular beacons by the trans-cleavage activity to produce the detection signal. To improve sensitivity, nucleic acid amplification technologies are usually introduced to give a pre-amplification of the nucleic acid targets, increasing the detection sensitivity extraordinarily. Polymerase chain reaction (PCR) technology has been used for pre-amplification in laboratories, and isothermal amplification technologies are applied to meet point-of-care testing (POCT) needs because they avoid the use of sophisticated thermal cycling devices. The recombinase polymerase amplification (RPA) technology that amplifies nucleic acid targets isothermally at 37–42°C has been combined with CRISPR-Cas nucleases to establish advantageous nucleic acid detection assays, e.g., the SHERLOCK, which combines with Cas13a, and the DETECTR, which combines with Cas12a. It has been challenging to integrate Cas nucleases and RPA in a one-pot reaction system because the cleavage activity of Cas stimulated by even small amounts of the targets can interfere with amplification by digesting the primers or the newly amplified fragments. Thus, many assays based on Cas nucleases and RPA are in a two-step setting, with pre-amplification and Cas cleavage being isolated as two independent procedures. The two-step setting ensures that amplification and cleavage occur under favorable conditions but sacrifices operational convenience and introduces the risk of cross-contamination. In efforts to establish one-pot RPA-Cas assays, many strategies have been applied, including the use of photocontrolled guide RNA to activate the Cas nuclease at a preferred timepoint, the use of a suboptimal protospacer adjacent motif (PAM) to suppress Cas activity, the generation of dynamic aqueous multiphase with sucrose or glycerol to partially separate the two reactions, and extensive optimization of the RPA-CRISPR reaction system to achieve a subtle balance between the two reactions. In this study, a one-pot CRISPR-Cas12b and RPA combined assay with a temperature switch (CRATS) was established utilizing the reaction temperature difference between RPA and CRISPR-Cas12b cleavage. The Cas12b used in this study, AaCas12b, is a type V-B CRISPR‒Cas nuclease from Alicyclobacillus acidiphilus. It has a bi-lobed architecture consisting of an α-helical recognition lobe containing the REC domains and a nuclease lobe containing the WED, RuvC and Nuc domains. As a dual-RNA-guided DNA endonuclease, Cas12b can be guided by a chimeric single-guide (sg) RNA, and its trans-cleavage activity is specifically activated by the DNA target and results in nonspecific cleavage of single-stranded (ss) DNA molecules, which can be used to produce detection signals if the ssDNA is appropriately labelled as the molecular beacon. As the temperature for RPA is 37°C and the trans-cleavage of Cas12b is active at 60°C, CRATS uses temperature switching to adjust the major on-going reaction in the one-pot system and realizes sequential amplification of the target and cleavage reactions for signal detection. The detection target of this study, monkeypox virus, is an infectious pathogen that has caused the announcement of the Public Health Emergency of International Concern (PHEIC) by the World Health Organization (WHO) twice in recent years. In this one-pot CRATS assay, the reaction reagents of CRISPR-Cas12b and RPA are mixed in a single tube. After the addition of the sample containing the detection target, the reaction was carried out at 37°C for 20 min for amplification, followed by 60°C for 20 min for Cas12b cleavage. The fluorescently labelled molecular beacon is cleaved by Cas12b to release the FAM fluorophore from quenching, producing a fluorescence signal that is visualized under blue light. CRATS shows a high sensitivity of 100 copies of the target DNA per reaction and good specificity, providing a novel strategy of temperature switching to integrate CRISPR-Cas and RPA in a one-pot reaction system. Moreover, it provides a POCT-friendly tool for the detection of the important infectious pathogen monkeypox virus.

1. Introduction

CRISPR-Cas nucleases have been extensively used in molecular detections, especially highly sensitive nucleic acid detections. In these detections, Cas nucleases are programmed by the guide RNA to respond to the detection targets and cleave the chemically labelled molecular beacons by the trans-cleavage activity to produce the detection signal. To improve sensitivity, nucleic acid amplification technologies are usually introduced to give a pre-amplification of the nucleic acid targets, increasing the detection sensitivity extraordinarily. Polymerase chain reaction (PCR) technology has been used for pre-amplification in laboratories, and isothermal amplification technologies are applied to meet point-of-care testing (POCT) needs because they avoid the use of sophisticated thermal cycling devices. The recombinase polymerase amplification (RPA) technology that amplifies nucleic acid targets isothermally at 37–42°C has been combined with CRISPR-Cas nucleases to establish advantageous nucleic acid detection assays, e.g., the SHERLOCK, which combines with Cas13a, and the DETECTR, which combines with Cas12a.

It has been challenging to integrate Cas nucleases and RPA in a one-pot reaction system because the cleavage activity of Cas stimulated by even small amounts of the targets can interfere with amplification by digesting the primers or the newly amplified fragments. Thus, many assays based on Cas nucleases and RPA are in a two-step setting, with pre-amplification and Cas cleavage being isolated as two independent procedures. The two-step setting ensures that amplification and cleavage occur under favorable conditions but sacrifices operational convenience and introduces the risk of cross-contamination. In efforts to establish one-pot RPA-Cas assays, many strategies have been applied, including the use of photocontrolled guide RNA to activate the Cas nuclease at a preferred timepoint, the use of a suboptimal protospacer adjacent motif (PAM) to suppress Cas activity, the generation of dynamic aqueous multiphase with sucrose or glycerol to partially separate the two reactions, and extensive optimization of the RPA-CRISPR reaction system to achieve a subtle balance between the two reactions.