Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance
The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.
Authors
- Xiao‐Yong Fan (ORCID: https://orcid.org/0000-0001-6654-3559)
- Guoping Zhao (ORCID: https://orcid.org/0000-0002-7621-6620)
- Huan Huang (ORCID: https://orcid.org/0000-0002-5795-6922)
- Christopher J. Lyon (ORCID: https://orcid.org/0000-0003-2319-2933)
- Yajuan Dong
- Shuai Ding (ORCID: https://orcid.org/0000-0003-3108-9434)
- Zhen Huang (ORCID: https://orcid.org/0000-0002-3990-7350)
- Yun Peng (ORCID: https://orcid.org/0000-0002-8163-267X)
- Ganggang Zhang (ORCID: https://orcid.org/0000-0002-6775-163X)
- Jin Wang (ORCID: https://orcid.org/0000-0003-4008-9284)
- Yang Liu (ORCID: https://orcid.org/0000-0002-5221-7725)
- Yang Yang (ORCID: https://orcid.org/0000-0002-6489-4459)
- Shuihua Lu
- Fuxiang Wang
- Ye Hu (ORCID: https://orcid.org/0000-0001-7255-5409)
- Chaosu Hu (ORCID: https://orcid.org/0000-0002-6503-0951)
- Xuchun Han
Institutions
- Tulane University (US)
- Huaibei Normal University (CN)
- WuXi AppTec (China) (CN)
- Phillips Exeter Academy (US)
- Southern University of Science and Technology (CN)
- National Clinical Research Center for Digestive Diseases (CN)
- Shanghai Public Health Clinical Center (CN)
- Jiangxi Academy of Sciences (CN)
- University of Chinese Academy of Sciences (CN)
- Shenzhen Third People’s Hospital (CN)
- Tsinghua University (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-11-21
- DOI
- https://doi.org/10.1038/s41467-025-65193-1
- Citations
- 2
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 1.61
Funders
- Basic and Applied Basic Research Foundation of Guangdong Province