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By Deliana InfanteReviewed by Danielle Ellis, B.Sc.

What is CRISPR diagnostics?
Emerging applications of CRISPR diagnostics
Notable CRISPR diagnostics platforms
Advantages and innovations in CRISPR diagnostics
The future of CRISPR diagnostics
References
Further reading

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CRISPR technology has revolutionized genetic testing and disease detection, offering precise, rapid, and cost-effective diagnostic solutions. This gene-editing tool has been adapted for molecular diagnostics, utilizing Cas proteins to detect specific nucleic acidsequences with high sensitivity. CRISPR-based platforms such as SHERLOCK and DETECTR enable point-of-care testing, multiplexed detection, and portable diagnostics, transforming healthcare delivery in resource-limited settings.

What is CRISPR diagnostics?

CRISPR technology, originally discovered as a bacterial defense mechanism against viruses, has revolutionized genetic engineering and is rapidly transforming the field of diagnostics.1

These systems consist of clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins.1  CRISPR-Cas systems utilize RNA guides to direct Cas proteins to specific DNA sequences, where the Cas protein cleaves the DNA. This ability to precisely target and cut DNA is the foundation for gene editing applications.1  

Understanding CRISPR Therapy

Its ability to recognize and bind to specific nucleic acid sequences with high sensitivity and specificity makes it ideal for diagnostic applications.2 A guide RNA (gRNA) is designed to be complementary to the target nucleic acid sequence (DNA or RNA) of the pathogen or biomarker of interest.2  

When the gRNA finds and binds to its target, it activates the Cas enzyme.2 Upon activation, some Cas enzymes exhibit “collateral cleavage” activity, where they cleave nearby reporter molecules.2 This generates a detectable diagnostic signal, indicating the presence of the target sequence.2  

Noteworthy among the recent advancements are SHERLOCK (Specific High-sensitivity Enzymatic Reporter unlocking)3,4 and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter)4.

Image Credit: Panuwach/Shutterstock.com

Emerging applications of CRISPR diagnostics

CRISPR diagnostics are being developed for a wide range of diseases, showcasing its potential for early and precise detection. 

SHERLOCK and DETECTR were rapidly adapted for SARS-CoV-2 detection, offering rapid and accurate results5,6. Studies demonstrated high sensitivity and specificity, comparable to PCR, with potential for point-of-care use.5,7

CRISPR-based assays have shown promise in the diagnosis of different pathogens infections, such as Mycobacterium tuberculosis.8 They also have been successfully used to detect different Plasmodium species in patient blood samples, offering a new and powerful tool for malaria diagnosis.9

In addition to its application in infectious diseases, there are several CRISPR-based developments for the diagnosis of other diseases, including cancer, where it is being used to detect cancer-specific biomarkers in circulating tumor DNA (ctDNA), enabling early detection and monitoring of cancer.10,11 In particular, rapid and portable cancer diagnostic tools are being developed for use in clinical settings.10,11

Notable CRISPR diagnostics platforms 

SHERLOCK and DETECTR, two leading CRISPR-based diagnostic platforms, have garnered significant attention due to their potential to revolutionize disease detection.  

In 2019, SHERLOCK technology was developed at the Broad Institute of MIT and Harvard by a team of renowned scientists and entrepreneurs12; in 2024, Sherlock Biosciences, the company behind its commercialization, was acquired by OraSure Technologies.13 On the other hand, DETECTR was pioneered by Jennifer Doudna and her colleagues at the UC Berkeley as part of their work on CRISPR systems, finally funding Mammoth Biosciences.14

While SHERLOCK and DETECTR are distinct technologies, they share several important connections that highlight their common foundations and potential for complementary applications.5,15

Both technologies are based on the CRISPR-Cas system but utilize different CRISPR-associated proteins, Cas13 for SHERLOCK and Cas12a for DETECTR, to achieve their highly specific diagnostic capabilities.5,15 A key feature of both technologies is their reliance on the collateral cleavage activity of these Cas proteins, which generates detectable signals upon target recognition.5,15

Additionally, both SHERLOCK and DETECTR often incorporate isothermal amplification techniques, such as recombinase polymerase amplification (RPA), to enhance the sensitivity of detection, making them effective for rapid diagnostics.5,15

Notably, researchers have developed SHERLOCK and DETECTR assays that can simultaneously detect multiple targets, allowing for comprehensive testing in a single reaction.11,16 Their applications are similarly versatile, ranging from the detection of viral infections like SARS-CoV-2 to distinguishing between closely related pathogens or specific disease biomarkers, showing their utility in molecular diagnostics.11,16

Advantages and innovations in CRISPR diagnostics

Beyond multiplexing capabilities of the already established CRISPR-based diagnostic systems, researchers have developed a powerful search algorithm that has identified 188 previously unknown CRISPR-associated gene modules, expanding the known diversity of CRISPR systems and their associated functions.17

This discovery significantly expands the CRISPR toolbox, offering the potential for more precise gene editing and diagnostics with fewer off-target effects in the near future.17

Science explains: CRISPR diagnostics

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Additionally, in 2024, Sherlock Biosciences started a clinical trial for a rapid, over-the-counter (OTC) diagnostic test for Chlamydia and Gonorrhea.18 This molecular test analyzes self-collected swabs and provides results in under 30 minutes.18

The PROMISE trial compares the test to the current gold standard, PCR, with 2,500 participants.18 Sherlock aims to obtain FDA approval for OTC use, making STI testing more accessible, particularly for underserved populations. This user-friendly test could revolutionize STI diagnosis and management by enabling rapid detection and treatment.18

The future of CRISPR diagnostics

CRISPR-based diagnostics hold immense promise for revolutionizing healthcare, but their widespread adoption faces several challenges.19 Technical limitations remain, including minimizing off-target effects and ensuring high sensitivity across diverse applications.19

Despite these challenges, exciting developments are emerging, such as integrating CRISPR diagnostics with machine learning to enhance accuracy and automate analysis.20Multiplexed detection, which allows simultaneous screening for multiple diseases or genetic markers, also offers comprehensive diagnostic capabilities.16  

In the future, CRISPR diagnostics have the potential to transform the field of personalized medicine. By enabling the rapid and precise identification of genetic predispositions, these tools can guide tailored treatment strategies and preventive measures. Furthermore, real-time disease monitoring could become a reality, allowing for the early detection and intervention of medical conditions.

References

1. Chhipa, A. S., Radadiya, E. & Patel, S. CRISPR-Cas based diagnostic tools: Bringing diagnosis out of labs. Diagn Microbiol Infect Dis 109, 116252 (2024). https://doi.org/10.1016/j.diagmicrobio.2024.116252
2. Lee, H. Y. et al. CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection. Anal Chem (2024). https://doi.org/10.1021/acs.analchem.4c01246
3. Sherlock Biosciences, <https://sherlock.bio/> (n.d.).
4. Mustafa, M. I. & Makhawi, A. M. SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases. J Clin Microbiol 59(2021). https://doi.org/10.1128/JCM.00745-20
5. Broughton, J. P. et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol38, 870-874 (2020). https://doi.org/10.1038/s41587-020-0513-4
6. Biosciences, S. Sherlock Biosciences Receives FDA Emergency Use Authorization for CRISPR SARS-CoV-2 Rapid Diagnostic, <https://sherlock.bio/sherlock-biosciences-receives-fda-emergency-use-authorization-for-crispr-sars-cov-2-rapid-diagnostic/> (2020).
7. Biosciences, S. Sherlock Biosciences Announces Clinical Data from Dartmouth-Hitchcock Health’s Pilot Study of Sherlock™ CRISPR SARS-CoV-2 Kit. (2020).
8. Zhang, X. et al. A new method for the detection of Mycobacterium tuberculosis based on the CRISPR/Cas system. BMC Infect Dis 23, 680 (2023). https://doi.org/10.1186/s12879-023-08656-4
9. Cunningham, C. H. et al. A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, species differentiation, and drug-resistance genotyping. EBioMedicine 68, 103415 (2021). https://doi.org/10.1016/j.ebiom.2021.103415
10. S., S., V., C., D., K. P., P., N. & V., S. CRISPR based biosensing: An ultrasensitive theranostic tool for the detection of early Breast Cancer biomarkers – A mini review Biosensors and Bioelectronics: X 14 (2023). https://doi.org/https://doi.org/10.1016/j.biosx.2023.100367
11. Hao, L. et al. CRISPR-Cas-amplified urinary biomarkers for multiplexed and portable cancer diagnostics. Nat Nanotechnol 18, 798-807 (2023). https://doi.org/10.1038/s41565-023-01372-9
12. B., B. Sherlock Biosciences licenses Wyss Institute’s ambient nucleic acid amplification technology from Harvard to develop highly accurate, low-cost diagnostics for point-of-need. Wyss Institute (2022).
13. Technologies, O. OraSure Technologies Acquires Sherlock Biosciences, <https://orasure.gcs-web.com/news-releases/news-release-details/orasure-technologies-acquires-sherlock-biosciences> (2024).
14. Biosciences, M. <https://mammoth.bio/> (n.d.).
15. 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). https://doi.org/10.1038/s41596-019-0210-2
16. Pena, J. M. et al. Real-Time, Multiplexed SHERLOCK for in Vitro Diagnostics. J Mol Diagn 25, 428-437 (2023). https://doi.org/10.1016/j.jmoldx.2023.03.009
17. Altae-Tran, H. et al. Uncovering the functional diversity of rare CRISPR-Cas systems with deep terascale clustering. Science 382, eadi1910 (2023). https://doi.org/10.1126/science.adi1910
18. D., P. Sherlock starts clinical trial for rapid OTC diagnostics for STIs, <https://www.clinicaltrialsarena.com/news/sherlock-starts-clinical-trial-for-rapid-otc-diagnostics-for-stis/> (2024).
19. Ghouneimy, A., Mahas, A., Marsic, T., Aman, R. & Mahfouz, M. CRISPR-Based Diagnostics: Challenges and Potential Solutions toward Point-of-Care Applications. ACS Synth Biol 12, 1-16 (2023). https://doi.org/10.1021/acssynbio.2c00496
20. Dixit, S., Kumar, A., Srinivasan, K., Vincent, P. & Ramu Krishnan, N. Advancing genome editing with artificial intelligence: opportunities, challenges, and future directions. Front Bioeng Biotechnol 11, 1335901 (2023). https://doi.org/10.3389/fbioe.2023.1335901