Three innovative technologies utilizing CRISPR-Cas13, Cas12a, and Cas9 demonstrate how CRISPR might be used for more than gene editing, while highlighting potential to develop new diagnostics for both the medical laboratory and point-of-care (POC) testing markets
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is in the news again! The remarkable genetic-editing technology is at the core of several important developments in clinical laboratory and anatomic pathology diagnostics, which Dark Daily has covered in detail for years.
Now, scientists at three universities are investigating ways to expand CRISPR’s use. They are using CRISPR to develop new diagnostic tests, or to enhance the sensitivity of existing DNA tests.
One such advancement improves the sensitivity of SHERLOCK (Specific High Sensitivity Reporter unLOCKing), a CRISPR-based diagnostic tool developed by a team at MIT. The new development harnesses the DNA slicing traits of CRISPR to adapt it as a multifunctional tool capable of acting as a biosensor. This has resulted in a paper-strip test, much like a pregnancy test, that can that can “display test results for a single genetic signature,” according to MIT News.
Such a medical laboratory test would be highly useful during pandemics and in rural environments that lack critical resources, such as electricity and clean water.
One Hundred Times More Sensitive Medical Laboratory Tests!
Co-lead authors Jonathan Gootenberg, PhD Candidate, Harvard University and Broad Institute; and Omar Abudayyeh, PhD and MD student, MIT, published their findings in Science. They used CRISPR Cas13 and Cas12a to chop up RNA in a sample and RNA-guided DNA binding to target genetic sequences. Presence of targeted sequences is then indicated using a paper-based testing strip like those used in consumer pregnancy tests.
MIT News highlighted the high specificity and ease-of-use of their system in detecting Zika and Dengue viruses simultaneously. However, researchers stated that the system can target any genetic sequence. “With the original SHERLOCK, we were detecting a single molecule in a microliter, but now we can achieve 100-fold greater sensitivity … That’s especially important for applications like detecting cell-free tumor DNA in blood samples, where the concentration of your target might be extremely low,” noted Abudayyeh.
Creating a Cellular “Black Box” using CRISPR
“The [CRISPR] technology demonstrates potential for many healthcare applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer,” stated senior author Feng Zhang, PhD. Zhang, shown above in the MIT lab named after him, is a Core Institute Member of the Broad Institute, Associate Professor in the departments of Brain and Cognitive Sciences and Biological Engineering at MIT, and a pioneer in the development of CRISPR gene-editing tools. (Photo copyright: MIT.)
Another unique use of CRISPR technology involved researchers David Liu, PhD, and Weixin Tang, PhD, of Harvard University and Howard Hughes Medical Institute (HHMI). Working in the Feng Zhang laboratory at the Broad Institute, they developed a sort of “data recorder” that records events as CRISPR-Cas9 is used to remove portions of a cell’s DNA.
They published the results of their development of CRISPR-mediated analog multi-event recording apparatus (CAMERA) systems, in Science. The story was also covered by STAT.
“The order of stimuli can be recorded through an overlapping guide RNA design and memories can be erased and re-recorded over multiple cycles,” the researchers noted. “CAMERA systems serve as ‘cell data recorders’ that write a history of endogenous or exogenous signaling events into permanent DNA sequence modifications in living cells.”
This creates a system much like the “black box” recorders in aircraft. However, using Cas9, data is recorded at the cellular level. “There are a lot of questions in cell biology where you’d like to know a cell’s history,” Liu told STAT.
While researchers acknowledge that any medical applications are in the far future, the technology holds the potential to capture and replay activity on the cellular level—a potentially powerful tool for oncologists, pathologists, and other medical specialists.
Using CRISPR to Detect Viruses and Infectious Diseases
Another recently developed technology—DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR)—shows even greater promise for utility to anatomic pathology groups and clinical laboratories.
Also recently debuted in Science, the DETECTR system is a product of Jennifer Doudna, PhD, and a team of researchers at the University of California Berkeley and HHMI. It uses CRISPR-Cas12a’s indiscriminate single-stranded DNA cleaving as a biosensor to detect different human papillomaviruses (HPVs). Once detected, it signals to indicate the presence of HPV in human cells.
Despite the current focus on HPVs, the researchers told Gizmodo they believe the same methods could identify other viral or bacterial infections, detect cancer biomarkers, and uncover chromosomal abnormalities.
Future Impact on Clinical Laboratories of CRISPR-based Diagnostics
Each of these new methods highlights the abilities of CRISPR both as a data generation tool and a biosensor. While still in the research phases, they offer yet another possibility of improving efficiency, targeting specific diseases and pathogens, and creating new assays and diagnostics to expand medical laboratory testing menus and power the precision medicine treatments of the future.
As CRISPR-based diagnostics mature, medical laboratory directors might find that new capabilities and assays featuring these technologies offer new avenues for remaining competitive and maintaining margins.
However, as SHERLOCK demonstrates, it also highlights the push for tests that produce results with high-specificity, but which do not require specialized medical laboratory training and expensive hardware to read. Similar approaches could power the next generation of POC tests, which certainly would affect the volume, and therefore the revenue, of independent clinical laboratories and hospital/health system core laboratories.
Multiplexed and Portable Nucleic Acid Detection Platform with Cas13, Cas12a, and Csm6
Rewritable Multi-Event Analog Recording in Bacterial and Mammalian Cells
CRISPR-Cas12a Target Binding Unleashes Indiscriminate Single-Stranded DNase Activity
Researchers Advance CRISPR-Based Tool for Diagnosing Disease
CRISPR Isn’t Just for Gene Editing Anymore
CRISPR’s Pioneers Find a Way to Use It as a Glowing Virus Detector
With New CRISPR Inventions, Its Pioneers Say, You Ain’t Seen Nothin’ Yet
New CRISPR Tools Can Detect Infections Like HPV, Dengue, and Zika
Breakthrough DNA Editing Tool May Help Pathologists Develop New Diagnostic Approaches to Identify and Treat the Underlying Causes of Diseases at the Genetic Level
CRISPR-Related Tool Set to Fundamentally Change Clinical Laboratory Diagnostics, Especially in Rural and Remote Locations
Harvard Researchers Demonstrate a New Method to Deliver Gene-editing Proteins into Cells: Possibly Creating a New Diagnostic Opportunity for Pathologists
SHERLOCK makes accurate, fast diagnoses for about 61-cents per test with no refrigeration needed; could give medical laboratories a new diagnostic tool
Genetics researchers have been riveted by ongoing discoveries related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) for some time now and so have anatomic pathology laboratories. The diagnostic possibilities inherent in CRISPR have been established, and now, a new diagnostic tool that works with CRISPR is set to change clinical laboratory diagnostics in a foundational way.
The tool is called SHERLOCK, which stands for (Specific High-sensitivity Enzymatic Reporter unLOCKing). And it is causing excitement in the scientific community for several reasons:
- It can detect pathogens in extremely small amounts of genetic matter;
- Tests can be performed using urine and/or saliva rather than blood;
- The tests are extremely sensitive; and they
- Cost far less than the diagnostic tests currently in use.
In an article published in Science, researchers described SHERLOCK tests that can distinguish between strains of Zika and Dengue fever, as well as determining the difference between mutations in cell-free tumor DNA.
How SHERLOCK and CRISPR Differ and Why That’s Important
Scientists have long suspected that CRISPR could be used to detect viruses. However, far more attention has been given to the its genome editing capabilities. And, there are significant differences between how CRISPR and SHERLOCK work. According to the Science article, when CRISPR is used to edit genes, a small strip of RNA directs an enzyme capable of cutting DNA to a precise location within a genome. The enzyme that CRISPR uses is called Cas9 (CRISPR associated protein 9). It works like scissors, snipping the strand of DNA, so that it is either damaged or replaced by a healthy, new sequence.
SHERLOCK, however, uses a different enzyme—Cas13a (originally dubbed C2c2 by the researchers who discovered it). Cas13a goes to RNA, rather than DNA, and once it starts cutting, it doesn’t stop. It chops through any RNA it encounters. The researchers who developed SHERLOCK describe these cuts as “collateral cleavage.” According to an article published by STAT, “All that chopping generates a fluorescent signal that can be detected with a $200 device or, sometimes, with the naked eye.”
The screenshot above is from a video in which Feng Zhang, PhD (center), a Core Member of the Broad Institute at MIT and one of the lead researchers working on SHERLOCK, and his research team, explain the difference and value SHERLOCK will make in the detection of diseases like Zika. Click on the image above to watch the video. (Video copyright: Broad Institute/MIT.)
Early Stage Detection in Clinical Laboratories
A research paper published in Science states that SHERLOCK can provide “rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity.” Attomolar equates to about one part per quintillion—a billion-billion. According to the article on the topic also published in Science, “The detection sensitivity of the new CRISPR-Cas13a system for specific genetic material is one million times better than the most commonly used diagnostic technique.” Such sensitivity suggests that clinical laboratories could detect pathogens at earlier stages using SHERLOCK.
The Stat article notes that, along with sensitivity, SHERLOCK has specificity. It can detect a difference of a single nucleotide, such as the difference between the African and Asian strains of Zika (for example, the African strain has been shown to cause microcephaly, whereas the Asian strain does not). Thus, the combination of sensitivity and specificity could mean that SHERLOCK would be more accurate and faster than other diagnostic tests.
Clinicians in Remote Locations Could Diagnose and Treat Illness More Quickly
Perhaps one of the most important aspects of SHERLOCK is the portability and durability of the test. It can be performed on glass fiber paper and works even after the components have been freeze dried. “We showed that this system is very stable, so you can really put it on a piece of paper and it will survive. You don’t have to refrigerate it all the times,” stated Feng Zhang, PhD, in an interview with the Washington Post. Zhang is a Core Member of the Broad Institute at MIT and was one of the scientists who developed CRISPR.
The researchers note that SHERLOCK could cost as little as 61-cents per test to perform. For clinicians working in remote locations with little or no power, such a test could improve their ability to diagnose and treatment illness in the field and possibly save lives.
“If you had something that could be used as a screening test, very inexpensively and rapidly, that would be a huge advance, particularly if it could detect an array of agents,” stated William Schaffner, MD, Professor and Chair of the Department of Preventive Medicine at Vanderbilt University Medical Center, in the Post article. Schaffner describes the Broad Institute’s research as being “very, very provocative.”
The test could radically change the delivery of care in more modern settings, as well. “It looks like one significant step on the pathway [that] is the Holy Grail, which is developing point-of-care, or bedside detection, [that] doesn’t require expensive equipment or even reliable power,” noted Scott Weaver, PhD, in an article on Big Think. Weaver is a Professor and Director at the Institute for Human Infections and Immunity University of Texas Medical Branch in Galveston, Texas.
Just the Beginning
Anatomic pathologists and clinical laboratories will want to follow SHERLOCK’s development. It could be on the path to fundamentally transforming the way disease gets diagnosed in their laboratories and in the field.
According to the Post article, “The scientists have filed several US patent applications on SHERLOCK, including for uses in detecting viruses, bacteria, and cancer-causing mutations.” In addition to taking steps to secure patents on the technology, the researchers are exploring ways to commercialize their work, as well as discussing the possibility of launching a startup. However, before this technology can be used in medical laboratory testing, SHERLOCK will have to undergo the regulatory processes with various agencies, including applying for FDA approval.
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Trends in Genomic Research That Could Impact Clinical Laboratories and Anatomic Pathology Groups Very Soon
Pathologists and Clinical Laboratories May Soon Have a Test for Identifying Cardiac Patients at Risk from Specific Heart Drugs by Studying the Patients’ Own Heart Cells
Patent Dispute over CRISPR Gene-Editing Technology May Determine Who Will Be Paid Licensing Royalties by Medical Laboratories
U.S. Patent and Trademark Office will hold hearings to determine whether University of California Berkeley, or Broad Institute of Harvard and MIT, should receive patents for new genomic engineering technique
In the race to master gene-editing in ways that will advance genetic medicine and patient care, one of the hottest technologies is CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats. But now a patent fight has the potential to complicate how pathologists and other scientists use this exciting technology.
This dispute over the CRISPR patent—a tool that has been hailed as one of the biggest biotech breakthroughs of the decade—will likely be settled in the coming months by the United States Patent and Trademark Office (USPTO).
The USPTO will be reviewing key patents awarded for what is called CRISPR/Cas9. The technology is already generating novel therapies for diseases, which should create new opportunities for pathologists and medical laboratories. (more…)
This technology has potential to create a demand for pathologists to do genetic analysis as a companion diagnostic in support of physicians treating patients with gene-editing proteins
Researchers at Harvard University have demonstrated a new method to deliver gene-editing proteins into cells. This breakthrough could eventually trigger a demand for pathologists to do genetic analysis as the companion diagnostic needed to help clinicians select appropriate gene-editing therapies for their patients.
Of course, it will be several years before such a scenario is feasible. The related example are the companion diagnostic tests that clinical laboratories perform to guide a physician’s decision on an appropriate therapeutic drug. Continued development of gene-editing therapies has the potential to increase the need for pathologists and medical laboratory scientists to do genetic analysis as a companion diagnostic for patients who would benefit from a gene-editing therapy.
The Harvard University researchers used commercially available cationic lipids to deliver genome-editing proteins into cells. The system works on living animals and humans, and the technology enables scientists to precisely and easily change DNA sequences at exact locations. The full study was outlined in an October Nature Biotechnology article. (more…)