The Promise of CRISPR-Based Diagnostics

The Promise of CRISPR-Based Diagnostics

A conversation with Janice Chen, PhD, the co-founder and chief research officer of Mammoth Biosciences

Laura M. Bolt, PhD

Janice Chen received her PhD from the lab of Jennifer Doudna at University of California, Berkeley. She has authored multiple publications and patents related to CRISPR mechanism and technologies. She also co-invented the programmable CRISPR-based detection technology called DETECTR. Janice was selected as a 2019 Forbes 30 Under 30 in Healthcare, and recently delivered a TEDx talk on the potential for CRISPR to democratize diagnostics.

Q: What is CRISPR and what medical applications does it have? 

A: CRISPR is an ancient bacterial immune system that harbors a programmable protein that can cut DNA or RNA. The system can be repurposed as a biological search engine because it uses an RNA molecule that guides the CRISPR protein to search through billions of sequences to find a matching nucleic acid target. The ability to harbor CRISPR proteins for gene editing, gene expression modulation, RNA editing, and DNA/RNA detection has enabled many applications in therapeutics and diagnostics. While CRISPR-based diagnostics are currently in development, the ability to detect and rewrite the underlying cause of genetic disease with CRISPR has huge medical potential.

Q: How can CRISPR be used to diagnose disease?

A: The finding that certain versions of CRISPR proteins can produce a realtime signal when they find a matching DNA or RNA sequence has enabled the use of CRISPR for detecting any disease with nucleic acid biomarkers. For example, CRISPR can be programmed to detect sequences from bacteria, viruses, or genetic mutations within our own cells. The researcher simply designs guide RNAs that allow CRISPR to specifically target a pathogen, such as Zika virus. Once the complex finds the presence of the viral RNA in a given sample, it cleaves a reporter molecule that releases a color change, indicating that the target is present.

Q: How does the use of CRISPR for diagnostics differ from its gene editing applications?

A: Both applications leverage the programmability of RNA-guided CRISPR proteins to search for a nucleic acid target. Unlike the well-known gene editing tool Cas9, other CRISPR proteins called Cas12, Cas13, and Cas14 used for diagnostics generate indiscriminate cleavage when the protein finds its matching target. This secondary activity was an unexpected mechanistic finding and is the part that enables CRISPR [to serve] as a detection tool, because these proteins have the ability to cleave a reporter molecule that produces a real-time signal only when the target is present.

Q: What research have you conducted in this area?

A: During my time as a graduate student in the Doudna Lab, my colleagues and I discovered a new activity for the Cas12 protein and showed that it could be repurposed to detect HPV in clinical samples. This detection activity has also been observed in other CRISPR protein families, namely Cas13 and Cas14, and there is active research on the underlying mechanisms of these CRISPR proteins and how they can be further developed for nucleic acid detection.

Q: What are some of the strengths and weaknesses of CRISPR in disease diagnostics?

A: There are several advantages of CRISPR-based diagnostics compared to traditional PCR-based methods for detection. These include high specificity due to enzymatic recognition of the target, fast turnaround times due to signal amplification, compatibility with low-cost form factors due to isothermal reactions, and rapid test development cycles due to the programmable system.

Given that CRISPR diagnostics is a new technology, it hasn’t undergone decades of development. We may be scratching only the surface of the possibilities of this technology. We envision several possibilities for CRISPRbased diagnostic products—reagent kits, integration onto existing bench-top equipment, and fully integrated devices for point-of-care or at-home use. Each class of product will have varying degrees of technical, safety, and regulatory requirements, but the underlying goal will be to improve on existing technologies and enable greater access to diagnostic information. In the case of at-home use, there are potential ethical challenges regarding data sharing and concerns around the need for clinical counseling upon receiving a test result. We’re currently having discussions about how to ensure patient privacy and appropriate counseling.

Q: What has been the role of Mammoth Biosciences in advancing the use of CRISPR in disease diagnostics?

A: Mammoth is the first company to take the steps toward commercializing CRISPR diagnostics. We are leveraging our expanding toolbox of CRISPR proteins with increased performance features to enable robust DNA and RNA detection. We are also developing the platform that enables rapid design, validation, and prototyping of CRISPRbased diagnostic tests that can be widely used for any nucleic acid biomarker in a simple, low-cost form factor. We think the simplicity and accuracy of CRISPR-based diagnostics will support increased accessibility of heath information, early disease detection, clinical actionability, and personalized medicine.

Q: What do you see happening in CRISPR-based diagnostics in the future?

A: Despite CRISPR diagnostics being a new technology, we are experiencing a significant demand for solutions related to improvements to existing technologies and diagnostic tests that can be performed at point-of-need. We are also observing trends toward personalized medicine and decentralization of healthcare, which will likely increase as hardware, software, and molecular technologies continue to advance. Although CRISPR diagnostics is one link in the larger value chain of decentralized healthcare, it addresses an unmet need for rapid and sensitive diagnostics in the clinic, in the field, and at home.

DETECTR: A Simple Molecular Diagnostics Platform

Last year, Chen and colleagues published a paper in Science on a newly discovered behavior of the CRISPR-Cas12a protein. Like Cas9, Cas12a has the ability to generate targeted, double-stranded DNA breaks. The researchers found that binding and cutting of a targeted double-stranded DNA sequence unleashes indiscriminate cutting of singlestranded DNA by Cas12a. They applied this discovery to develop a platform they dubbed DNA endonuclease targeted CRISPR trans reporter, or DETECTR, which combines Cas12a, its guide RNA, a fluorescent reporter molecule, and recombinase polymerase amplification in a single reaction. DETECTR enabled rapid and accurate detection of cervical cancer-associated human papillomavirus types 16 and 18 in patient samples, the authors reported, thus demonstrating its potential application as a simple molecular diagnostics platform.