Development of a Rapid and Sensitive CasRx-Based Diagnostic Assay for SARS-CoV-2
Abstract
The development of an extensive toolkit for potential point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect SARS-CoV-2. Herein, we outline the development of an alternative CRISPR nucleic acid diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens XPD3002 (CasRx) to detect SARS-CoV-2, an approach we term SENSR (sensitive enzymatic nucleic acid sequence reporter) that can detect attomolar concentrations of SARS-CoV-2. We demonstrate 100% sensitivity in patient-derived samples by lateral flow and fluorescence readout with a detection limit of 45 copy/μL. This technology expands the available nucleic acid diagnostic toolkit, which can be adapted to combat future pandemics.
Additional Information
© 2021 American Chemical Society. This article is made available via the ACS COVID-19 subset for unrestricted RESEARCH re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. Received: May 26, 2021; Accepted: October 14, 2021; Published: October 29, 2021. We thank the SEARCH (San Diego Epidemiology and Research for COVID-19 Health) Alliance for providing clinical samples. This work was supported in part by UCSD Seed Funds for Emergent COVID-19 Related Research, a Directors New Innovator award from NIH/NIAID (DP2 AI152071-01), NIH/NIAID R21 (1R21AI149161), and a DARPA Safe Genes Program Grant (HR0011-17-2-0047) awarded to O.S.A., and a Director's Pioneer Award from NCCIH (DP1 AT010885) to R.K., and the Molecular Biophysics Training Grant from NIH (T32 GM00832) to C.P.L., and the UCSD Return to Learn program via the EXCITE (EXpedited COVID-19 IdenTification Environment) lab. The views, opinions, and/or findings expressed should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. C.P.L. was supported by the Molecular Biophysics Training Grant, NIH Grant T32 GM00832. Author Contributions: D.J.B., D.C.-R., and C.P.L. are equal first author contributions. A.L.S. and T.Y. are equal second author contributions. O.S.A. conceptualized the study. D.J.B., D.C.-R., C.P.L., J.L., L.M.A., T.Y., I.A., and P.B.-F., performed, designed, and analyzed various molecular, bioinformatic, and protein biochemistry. A.L.S., R.R.R., R.K., and E.A.K. contributed to analyzing and designing experiments. All authors contributed to writing, analyzing the data, and approving the final manuscript. The authors declare the following competing financial interest(s): O.S.A. has a patent pending on this technology. All other authors declare no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described. Bioinformatic scripts can be found in File S1. A limited quantity of CasRx enzyme will be made available upon request.Attached Files
Published - acssensors.1c01088.pdf
Supplemental Material - se1c01088_si_001.xlsx
Supplemental Material - se1c01088_si_002.txt
Supplemental Material - se1c01088_si_003.mov
Supplemental Material - se1c01088_si_004.pdf
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Additional details
- PMCID
- PMC8577378
- Eprint ID
- 111721
- Resolver ID
- CaltechAUTHORS:20211102-222421621
- University of California, San Diego
- NIH
- DP2 AI152071-01
- NIH
- 1R21AI149161
- Defense Advanced Research Project Agency (DARPA)
- HR0011-17-2-0047
- NIH
- DP1 AT010885
- NIH Predoctoral Fellowship
- T32 GM00832
- Created
-
2021-11-02Created from EPrint's datestamp field
- Updated
-
2021-12-03Created from EPrint's last_modified field
- Caltech groups
- COVID-19