Next-Generation CRISPR Diagnostics with ENHANCEv2

Molecular diagnostic methods are still largely based on lab-based techniques such as qPCR and ELISA. CRISPR-Cas-based diagnostics offer several advantages and are moving clinical testing away from the lab and into the hands of the patients. Here we tell the story of the development and clinical validation of our ENHANCE and ENHANCEv2 platforms.

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Infectious diseases are as old as human civilization. Before our understanding of molecular biology, common diseases were thought to be caused by people being possessed by evil spirits or understood as acts of divine retribution. Now that we know better, we have realized that the culprit lies amongst the hordes of pathogenic viruses and bacteria that harm human health, rather than some malignant entity. Despite our progress, however, infectious diseases are becoming increasingly difficult to manage.

This is especially true for a pandemic where millions of people are infected simultaneously. Under such conditions, it becomes critical that laboratories are equipped to handle fluctuations and sudden surges in the demand for testing. Currently, many common human diseases are diagnosed using a method called real-time quantitative Polymerase Chain Reaction (qPCR). Although the qPCR technique is highly sensitive, accurate, and can help quantify pathogenic DNA, it suffers due to the need for expensive reagents, sophisticated equipment, and trained personnel, thereby limiting its use to a laboratory setting.

Still, all hope is not lost. Recent advances in molecular biology have revealed that hidden everywhere in nature are powerful biological tools whose potential scientists are only just beginning to unravel. Recently, one such tool, called CRISPR-Cas9, was discovered in bacteria and repurposed into a capable gene-editing technology with the potential to treat human diseases1. While Cas9 is the most renowned CRISPR-based enzyme, it isn’t the only one with applications for human health. Two new Cas enzymes, Cas12a and Cas13a, are now also being harnessed for the development of highly sensitive CRISPR-based diagnostics2.

CRISPR-based tools such as SHERLOCK3 and DETECTR4 outshine contemporary methods like RT-PCR by virtue of being rapid, inexpensive, and capable of being used at point-of-care. These tools utilize the nuclease property of the Cas12a and Cas13a enzymes and the specific targeting capability of the CRISPR RNA (crRNA) for the detection of pathogenic genes. More importantly, unlike other Cas enzymes, Cas12a and Cas13a are capable of non-specifically cleaving single-stranded DNA and RNA molecules, respectively, if the target pathogen is present in the clinical sample. It is by using this property, commonly termed as the trans-cleavage activity, that researchers have been able to develop CRISPR-based diagnostic assays that can be used at the point of care.

Fig.1: Workflow for the fluorescence-based and paper-strip-based ENHANCEv2 assay. Created with Biorender.com

Ongoing efforts in developing the next generation of CRISPR-based diagnostics have involved engineering the crRNA as well as the Cas enzymes to improve their overall diagnostic performance5–8. In the summer of 2019, our lab was tinkering with labeled crRNAs consisting of DNA linkers of different lengths at either their 3’-end or 5’-end. It was during this time that we made a rather serendipitous discovery with one of the control crRNAs. We observed that merely adding a short piece of 7-mer DNA to the 3’-end of the crRNA had the effect of greatly augmenting the rate of trans-cleavage activity of the LbCas12a enzyme, thus making it significantly faster than what it is naturally capable of. This modification also seemed to improve the specificity of CRISPR/Cas12a, thereby making it less prone to false-positive errors.

More importantly, we found the effects of the 7-mer DNA extension to the crRNA to be universal and spacer-independent, which meant that it could be added to any crRNA without affecting the fidelity of the CRISPR/Cas12a system or significantly affecting the cost of synthesis. We used this observation to develop ENHANCE9, a highly sensitive and specific diagnostic platform that was able to detect target pathogens down to femtomolar concentrations without any target amplification.

Apart from being the fastest known Cas12a based testing system, our ENHANCE platform was also the first CRISPR/Cas12a-based test that reported the detection of RNA by the formation of a DNA-RNA heteroduplex. We utilized ENHANCE for the detection of several clinically relevant pathogenic targets such as HIV and HCV. It was while our manuscript was still under internal review in the early months of 2020, that the COVID-19 pandemic swept through the globe and greatly disrupted all aspects of everyday life. The havoc caused by the pandemic created ripples in the supply-chain systems worldwide leading to a shortage of everything from toilet paper to diagnostic tests. We quickly jumped to action and designed an ENHANCE test targeting COVID-19. These results were subsequently incorporated in our initial papers9.

In this paper, we have provided clinical validation for our ENHANCE-based COVID-19 test using real-life patient nasal swabs. We have also developed ENHANCEv2, which is a further improvement on ENHANCE (Fig. 1). For this, we have combined the altered crRNA from the ENHANCE system with a mutated LbCas12a protein and a dual reporter capable of performing both fluorescence-based and lateral flow assays from a single step. We subjected the LbCas12a protein to the D156R mutation, which has previously been reported to improve its activity10. We observed that while the mutated protein by itself had a negligible effect on improving the speed of ENHANCE, the combined effect of the modified crRNA along with the mutated Cas12a showed a significant improvement in the trans-cleavage activity. This, in combination with the dual reporter, gave birth to ENHANCEv2.

Fig.2: Genomic architecture of the SARS-CoV-2 virus. Optimization of 6 different crRNAs against three 3 different genes, and samples obtained from different geographical regions. 

For this validation work, we first optimized several different crRNAs targeting gene segments within the nucleocapsid (N), envelope (E), and the RNA-dependent RNA polymerase (RdRP) regions of the SARS-CoV-2 viral genetic code (Fig.2). Using different crRNAs, we also performed inclusivity testing for samples obtained from different geographical regions such as Hong Kong, Italy, and the USA. In addition, exclusivity testing was performed against 31 other common bacteria and viruses to make sure that they would not interfere with the system and yield false positive detection. Through these experiments, we shortlisted two crRNAs that work robustly against samples from all geographic regions and show no cross-reactivity with other pathogens.

We then proceeded to use these two crRNAs for the clinical testing of 62 human patient samples for detection of the presence of SARS-CoV-2. Both ENHANCE and ENHANCEv2 showed 96.7% agreement with RT-qPCR results from the same clinical swabs using only 5 m of sample and a 20-minute CRISPR reaction time for the fluorescence-based test. The lateral flow assay agreed 100% with the fluorescence-based test results. 

Going a step further, we produced a lyophilized (or “freeze-dried”) version of ENHANCEv2, allowing for a CRISPR reaction time of as low as 3 minutes and the storage of the test at room temperature for several weeks while maintaining its detection abilities (Fig.3). While the lyophilization of CRISPR/Cas components was relatively straightforward, creating a complete lyophilized version of our test was a significant challenge because commercially available reagents for RT-LAMP, an important amplification step in our assay, were proprietary and contained ‘glycerol’ an ingredient that actively hinders the process of lyophilization. We eventually created our own RT-LAMP reagents in-house for the lyophilization process. Importantly, we validated the lyophilized version for all the 62 clinical samples that were previously tested, and the results were in agreement with the non-lyophilized version but took significantly less time to obtain.

Fig.3: Lyophilized version of ENHANCEv2 provides identical clinical performance but has enhanced speed and stability with multimodal detection.

We were interested in developing a lyophilized version for ENHANCEv2 because it offers several advantages. Firstly, the incorporation of the lyophilized version greatly assists with the ease of transportation of the test. Secondly, it provides accelerated reaction speed and improved stability at room temperature. Finally, it also simplifies the reaction setup as the reaction can be initiated by merely adding nuclease-free water and RT-LAMP amplified products to the lyophilized mix. Thus, by providing enhanced stability, speed, and ease of testing, ENHANCEv2 promotes large-scale manufacturing and enables easier deployment across the globe.  

Historically, the onset of infectious diseases has driven the development of diagnostic techniques, and the current pandemic has been no different. The field of CRISPR-based detection is still in its infancy, yet remarkable progress has been made since the advent of the pandemic. Molecular diagnostics is still largely dominated by qPCR and ELISA; however, CRISPR-based technologies are now able to expand that toolkit. The speed and low cost of these tests make them an attractive candidate for healthcare systems. We envision diagnostic tests to slowly move away from the lab and onto the field or point-of-care of the patient, and CRISPR-based tests will play a pivotal role in this process. 

You can read our paper here: https://doi.org/10.1038/s43856-021-00066-4

 References

  1. Sternberg, S. H. & Doudna, J. A. Expanding the Biologist’s Toolkit with CRISPR-Cas9. Mol. Cell 58, 568–574 (2015).
  2. Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F. & Collins, J. J. CRISPR-based diagnostics. Nat. Biomed. Eng. 5, 643–656 (2021).
  3. Kellner, M. J., Koob, J. G., Gootenberg, J. S., Abudayyeh, O. O. & Zhang, F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat. Protoc. 14, 2986–3012 (2019).
  4. Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439 (2018).
  5. Ooi, K. H. et al. An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing. Nat. Commun. 12, 1739 (2021).
  6. Kim, H. et al. Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide. Nucleic Acids Res. 48, 8601–8616 (2020).
  7. Shi Kai et al. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci. Adv. 7, eabc7802.
  8. Xiong, Y. et al. Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets. J. Am. Chem. Soc. 142, 207–213 (2020).
  9. Nguyen, L. T., Smith, B. M. & Jain, P. K. Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection. Nat. Commun. 11, 4906 (2020).
  10. Tóth, E. et al. Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Res. 48, 3722–3733 (2020).

Santosh Rananaware

Graduate Research Assistant, University of Florida