(236g) Invited Talk: Engineered CRISPR/Cas Systems As Rapid Diagnostics and Beyond

Introduction: Development of a rapid, sensitive, specific, inexpensive, easy-to-use, and accessible test kit for emerging infectious diseases that can detect active infections and can be implemented as a point-of-care (POC) diagnostic is desperately needed. Type V and VI CRISPR/Cas systems are emerging as next-generation of diagnostics. For example, two versions of CRISPR/Cas-based tests have been approved by the FDA under EUA for COVID-19 diagnostics^1,2. These CRISPR/Cas systems mediate a nonspecific collateral 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 a 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. We recently developed a range of engineered CRISPR/Cas12 systems towards developing a simple-to-use, one-pot, rapid and accurate clinical detection of SARS-CoV-2 and HCV RNA (Figure 1).

Materials and Methods: We recently explored combinatorial crRNA/Cas12a orthologs to test if crRNA from one bacterial species can function better with Cas12a from another species to expand the repertoire of CRISPR/Cas systems for diagnostics and clinically validated for COVID diagnosis (cCRISPR, medRxiv, 2021)³. We also investigated various thermophilic Cas12b systems to combine RT-LAMP and CRISPR at one-temperature to develop a simple one-pot test for detecting all five variants of concern and clinically validated it (SPADE, eBioMed.-The Lancet, 2022)⁴. By further engineering Cas12b enzymes using machine learning algorithms, including DeepDDG, we enhanced thermophilicity of BrCas12b and applied it to develop a more robust one-pot test that functions at 65-67^oC and demonstrated its application for SARS-CoV-2 and HCV RNA (SPLENDID, Cell Reports Medicine, pre-accepted, 2023)⁵. We also developed a split activator system that allows detection of a target RNA using a CRISPR/Cas12a (SAHARA, Nature Communications, under revision, 2023)⁶.

Results and Discussion: Several new functional combinations of crRNA/Cas12a orthologs (cCRISPR) were discovered and clinically validated for detecting SARS-CoV-2 in saliva, nasal swabs, and tracheal aspirates, achieving 97% accuracy³. We also identified a new thermophilic BrCas12b and developed a simple assay that combined target pre-amplification and CRISPR/Cas detection in one-pot discriminating all five variants of concern in saliva samples. This SPADE technology was successfully coupled with a point-of-care device and a also a phone-based setup for monitoring SARS-CoV-2 variants with 97% accuracy⁴. Finally, SPLENDID achieved robust detection of HCV RNA in serum samples with 91% accuracy. Other amplification-free methods such as SAHARA enable detection of shorter RNA targets such as miR155.

Conclusions: The engineered CRISPR-based approaches can be used to monitor and diagnose a number of pathogens in a point-of-care setting.

Acknowledgements: A part of this research has been funded in part by USISTEF/COVID-I/247/2020, FBCF- AGR00018466 (P.K.J.), CDC- U01GH002338, NIH-NIAID R21AI156321, NIH-NIAID R21AI168795, and NIH-NIGMS R35GM147788, the University of Florida, the UF Herbert Wertheim College of Engineering, and Dinesh O. Shah endowed professorship.

References: ^1. Chen JS, et al. Science. 2018; ^2. Gootenberg JS, et al. Science. 2017; ^3. Nguyen LT, et al. medRxiv. 2020; ^4. Nguyen LT, et al. eBioMed. 2022; ^5.Nguyen LT, et al. Cell Rep. Med.-accepted., 2023; ^6. Rananaware SR, et al. Nat. Comms.-in rev., 2023.