CRISPR/Cas12a Technology Combined with Recombinase Polymerase Amplification for Rapid and Portable Monkeypox Virus Detection

Abstract

Monkeypox virus (MPXV) continues to circulate in several countries in which it is not endemic, posing a potential threat to human health and world stability (1). Here, we developed a CRISPR/Cas12a approach combined with recombinase polymerase amplification (RPA) for MPXV testing within 40 min. Importantly, the two reaction systems were further integrated into a one-step fluorescence assay by optimizing the protospacer adjacent motif (PAM) sequence. The whole detection process can be completed within 30 min. In this study, when the RPA reaction mixture was placed immediately into the CRISPR/ Cas12a system, the Cas12a protein induced powerful nonspecific single-stranded DNA (ssDNA) transcleavage through specific CRISPR RNA (crRNA)-guided cleavage of doublestranded DNA (dsDNA) (2), which can be collected and analyzed by lateral flow strips or a portable fluorescence thermostatic amplifier (3) (Fig. 1A). We chose the F3L and B6R genes as targets for detection. Based on the newly published MPXV sequence (GenBank accession number ON563414.3), we designed specific crRNAs and found that they could be used to distinguish MPXV from variola viruses (VARVs) (see Fig. S1A and B in the supplemental material). To further test the specificity of the system, we also synthesized homologous fragments of other orthopoxviruses, including VARV E3L and B7R (GenBank accession number NC_001611.1), cowpox virus (CPXV) F3L and B4R (GenBank accession number X94355.2), and vaccinia virus (VACV) E3L and B5R (GenBank accession number LT966077.1). The detection system could not identify and amplify the target genes, therefore, Cas12a does not generate a cleavage signal, leading to negative results (Fig. 1B and C). Subsequently, we identified the limit of detection (LOD) for MPXV F3L as 10 copies/mL and that for B6R as 1 copy/mL (Fig. 1D). Compared with the real-time PCR (see Fig. S1C and D), our system improves detection sensitivity by a factor of 10. To test the ability of the system to cope with clinical samples, pseudotyped MPXVs containing the F3L and B6R fragments were obtained using Ad5 replication-defective adenovirus and diluted with throat swab and serum specimens. We obtained the same experimental results as described above (Fig. 1E and F). The lateral flow strip assay showed a sensitivity similar to that of the fluorescence thermostatic amplifier (Fig. 1G), and the test results were quantified (Fig. 1H). Recently, we read with great interest the article by Lu et al. (4), which combined the RPA and Cas12a complex in a one-step fluorescence assay. The assay adopted suboptimal PAMs (sPAMs) instead of canonical PAMs. Based on the research, we synthesized three crRNAs targeting sPAMs TTGG, TCTG, and ATTA (see Table S1). These crRNAs exhibited more sensitivity than TTTA in the one-pot reaction (see Fig. S1E). Because it had the shortest time of detection, TTGG was used for the following assay. Meanwhile, we designed a crRNA targeting the TTGG sequence of the B6R gene. The LOD reached 100 copies/mL (Fig. 1I and J). Although Editor Bo Zhang, Chinese Academy of Sciences Wuhan Institute of Virology Copyright © 2023 Li et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Tao Wang, wangtaobio@tju.edu.cn, or Yue Teng, yueteng@me.com. The authors declare no conflict of interest.