Engineered CRISPR/Cas systems as point-of-care diagnostics for infectious diseases and beyond

Long T. Nguyen^{1, †}, Santosh R. Rananaware^{1, †}, Lilia G. Yang^{1, †}, Nicolas C. Macaluso^{1, †, ††}, Julio E. Ocana-Ortiz², Katelyn S. Meister¹, Brianna L.M. Pizzano³, Luke Samuel W. Sandoval⁴, Raymond C. Hautamaki⁵, Zoe R. Fang⁵, Sara M. Joseph¹, Grace M. Shoemaker¹, Dylan R. Carman¹, Liwei Chang⁶, Noah R. Rakestraw⁷, Jon F. Zachary⁷, Sebastian Guerra⁸, Alberto Perez⁶, Piyush K. Jain^1,9,10,*

1Department of Chemical Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville FL, USA
2Department of Chemical Engineering, University of Puerto Rico – Mayagüez, Puerto Rico, USA
3Department of Agricultural and Biological Engineering, Herbert Wertheim College of Engineering, University of Florida, Gainesville FL, USA
4Department of Biology, College of Liberal Arts and Sciences, University of Florida, Gainesville, Florida, USA
5Department of Microbiology and Cell Science, College of Agricultural and Life Sciences, University of Florida, Gainesville, Florida, USA
6Department of Chemistry, College of Liberal Arts and Sciences, University of Florida, Gainesville, Florida, USA
7Department of Graduate Education, College of Medicine, University of Florida, Gainesville, Florida, USA
8Genetics Institute, College of Medicine, University of Florida, Gainesville, Florida, USA
9Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, Florida, USA
10UF Health Cancer Center, University of Florida, Gainesville FL, USA

Rapid, accurate, inexpensive, and easy-to-use tests that can detect infectious diseases in a point-of-care (POC) diagnostic are desperately needed. Type V and VI CRISPR/Cas systems have recently emerged as the next generation of diagnostics. For example, two versions of CRISPR/Cas-based tests (SHERLOCK and DETECTR) have been approved by the FDA under EUA for COVID-19 diagnostics. These CRISPR/Cas systems mediate a nonspecific collateral or ‘trans-cleavage’ of single-stranded DNA and RNA after recognizing their target DNA or RNA. This trans-cleavage activity has been widely exploited for nucleic acid detection by combining with fluorescence-based, paper-based, and electrochemical-based sensing technologies. However, these CRISPR/Cas systems are limited to picomolar detection unless the target is pre-amplified by isothermal amplification techniques such as RT-RPA or RT-LAMP. This adds additional steps and equipment, rendering them handicapped for use outside the lab. For example, the most commonly used RT-LAMP assay requires a temperature of 65C while the native CRISPR/Cas systems denature at that temperature. We recently engineered a highly thermophilic CRISPR/Cas12b system using machine learning algorithms to enhance their thermostability (Nguyen et al., Cell Reports Medicine, In Press, 2023)¹. This enabled single-pot detection of nucleic acids at higher temperatures improving the diagnostic capabilities of viral pathogens. This SPLENDID assay was clinically validated for detecting the Hepatitis C virus and SARS-CoV-2 RNA with high accuracy. The talk will cover various CRISPR-based diagnostic technologies from our lab^1-7  with a focus on recent developments^1,7.

References

Nguyen LT, Rananaware SR^§, Yang LG^§, Macaluso NC^§, Ocana-Ortiz JE, Meister KS, Pizzano BLM, Sandoval LSW, Hautamaki RC, Fang ZR, Joseph SM, Shoemaker GM, Carman DR, Chang L, Rakestraw NR, Zachary JF, Guerra S, Perez A, Jain PK, Engineering Highly Thermostable Cas12b via de novo Structural Analyses for One-Pot Detection of Nucleic Acids. Cell Reports Medicine (in press- May issue), 2023. SSRN: https://ssrn.com/abstract=4309028or http://dx.doi.org/10.2139/ssrn.4309028 Nguyen, L.N., Smith, B.M., & Jain P.K.* (2020) Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection. Nature Communications, https://doi.org/10.1038/s41467-020-18615-1. Nguyen, L.N., Gurijala, J., Rananaware S.R., Pizzano B.L.M., Brandon S.T., & Jain PK* (2021) CRISPR-ENHANCE: An enhanced nucleic acid detection platform using Cas12a. Methods, https://doi.org/10.1016/j.ymeth.2021.02.001. Nguyen L.N., Macalusa N.C., Jain, P.K.* (2021) A Combinatorial Approach towards Adaptability of 22 Functional Cas12a Orthologs for Nucleic Acid Detection in Clinical Samples. medRxiv https://doi.org/10.1101/2021.07.21.21260653. [Reviewed] Nguyen, L.N., Rananaware S.R., Pizzano B.L.M., Brandon S.T., & Jain P.K.* (2022) Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection. Communications Medicine- Nature, https://doi.org/10.1038/s43856-021-00066-4. Nguyen, L.N., Macaluso, N.C., Pizzano B.L.M., Cash, M., Spacek J., Karasek J., Dinglasan R.R., Salemi, M., & Jain P.K.* (2022) A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern. eBioMedicine – The Lancet, https://doi.org/10.1016/j.ebiom.2022.103926 Rananaware SR, Vesco EK, Shoemaker GM, Anekar SS, Sandoval LSW, Meister KS, Macaluso NC, Nguyen LT, and Jain, PK (2023) Programmable RNA detection with CRISPR-Cas12a. Nature Communications (in revision) Research square. 10.21203/rs.3.rs-2549171/v1. [Research Square][bioRxiv, 2023]