Rationally engineered CRISPR/Cas systems as diagnostics and beyond

Introduction:: Beyond the widely popular genome engineering applications, CRISPR/Cas systems are rapidly evolving as next-generation of diagnostics. There is an urgent need for the development of a rapid, accurate, inexpensive, easy-to-use, and accessible testing kit for infectious diseases that can be implemented as a point-of-care (POC) diagnostic. Type V and VI CRISPR/Cas systems have recently emerged as diagnostics for detecting SARS-CoV-2 RNA and have been approved by the FDA under EUA as COVID-19 diagnostics^1,2. These CRISPR/Cas systems mediate a nonspecific collateral trans-cleavage of random DNA and RNA after binding or cis-cleavage of 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, most CRISPR/Cas systems are limited to a picomolar detection limit unless the target is pre-amplified by methods such as RT-LAMP or RT-RPA. This necessitates additional steps and equipment, rendering them handicapped for use outside the high-complexity lab environment. We recently engineered
a range of CRISPR-based platform technologies towards developing a simple-to-use, rapid, and accurate detection of nucleic acids for infectious diseases^3-9. This presentation will include discovery of CRISPR/Cas systems (cCRISPR⁶ and SPADE⁷), engineering crRNAs to enhance activity (ENHANCE^3,4, SAHARA⁹), and engineering of Cas12b nuclease (ENHANCE v2⁵, SPLENDID⁸).

Materials and Methods:: We first engineered crRNAs with various length extensions with DNA or RNA on both 3’- and 5’-ends to understand and enhance the trans-cleavage activity (ENHANCE v1, Nature Communications, 2020; Methods, 2021)³ . Next, we combined ENHANCE v1 with engineered Cas12a and dual reporters, and then lyophilized the kits and clinically validated the system for detecting SARS-CoV-2 RNA using both fluorescence and lateral flow assays simultaneously (ENHANCE v2, Communications Medicine, 2022)⁵ . We also 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)⁶. We also investigated various thermophilic BrCas12b 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, eBioMedicine, 2022)⁷. We used a combination of structural design (AlphaFold and SWISS model) and deep learning tools (DeepDDG and HotSpot Wizard) to incorporate mutations that increase the thermostability of BrCas12b protein to achieve a single-pot RT-LAMP+CRISPR-based diagnostic test for HCV genotype 1 (GT1) and COVID-19 (SPLENDID, Cell Reports Medicine, 2023)⁸. Finally, we discovered that CRISPR/Cas12a orthologs that are widely known to detect only DNA targets can be tricked to recognize RNAs. We build a split activator system to detect RNAs based on CRISPR/Cas12a (SAHARA, Nature Communications-in rev., 2023)⁹.

Results, Conclusions, and Discussions:: With ENHANCE v1, we discovered that 7-nt DNA extensions on crRNAs can augment the trans-cleavage activity by 3.2-fold pushing the limit of detection to femtomolar range without any amplification³. ENHANCE v2 demonstrated high inclusivity and exclusivity and achieved 96.7% sensitivity and 96.7% specificity in clinical samples, satiability for at least 30 days at room temperature and enabled clinical diagnosis of COVID-19 with fluorescence and lateral flow assays from a single kit⁵. We identified several new functional combinations of crRNA/Cas12a orthologs (cCRISPR) and successfully validated them for COVID-19 diagnostics⁶. For developing our CRISPR-SPADE we identified a new thermophilic BrCas12b and developed a simple one-pot assay to detect all five variants of concern. We also developed a point-of-care device and a also a phone-based setup for monitoring SARS-CoV-2 variants (SPADE)⁷. Engineered BrCas12b allowed development of a simple one-pot diagnostic test for detecting HCV GT1 and SARS-CoV-2 RNA with high accuracy in clinical samples (SPLENDID)⁸. With SAHARA, we could detect native RNAs without amplification using CRISPR/Cas12a (SAHARA)⁹. In summary, we engineered various CRISPR-based approaches to detect RNAs more rapidly and accurately, opening doors for next-generation of point-of-care diagnostic tests.

Acknowledgements (Optional): : This research is funded in part by NIH-NIAID R21AI156321, NIH-NIAID R21AI168795, NIH-NIGMS R35GM147788, University of Florida, and Shah Foundation.

References (Optional): : 1. Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, https://doi.org/10.1126/science.aar6245.

2. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, https://doi.org/10.1126/science.aam9321.

3. 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.

4. 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.

5. 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.

6. 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]

7. 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

8. 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 (2023) Engineering Highly Thermostable Cas12b via de novo Structural Analyses for One-Pot Detection of Nucleic Acids. Cell Reports Medicine, https://doi.org/10.1016/j.xcrm.2023.101037.

9. 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.