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
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CRISPR’s Pioneers Find a Way to Use It as a Glowing Virus Detector
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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
Mobile point-of-care (POC) smartphone-based nucleic acid assay allows for quick turn arounds and accurate information in any healthcare setting, including resource limited and remote environments
DNA detection might soon be accomplished with the use of a smartphone. That’s the goal of a research effort at the University of California Los Angeles (UCLA). If this effort succeeds, it would give medical laboratories a new tool to use in genetic testing.
Clinical laboratory equipment is becoming more effective even as it shrinks in size and cost. One such device has been developed by Ozcan Laboratory Group, headed by UCLA professor Aydogan Ozcan, PhD. It is a portable, smartphone-based mobile lab with sensitivity and reliability on par with large-scale medical laboratory-based equipment.
Ozcan Lab’s portable DNA detection system, according to a UCLA press release, “leverages the sensors and optics of cellphones” and adapts them to read and report the presence of DNA molecules. The sensor uses a new detector dye mixture and reportedly produces a signal that is 10 to 20 times brighter than previous detector dye outputs.
This new system improves upon the optical detection abilities of current point-of-care nucleic acid tests (POCTs) and, according to a study published in the American Chemical Society’s ACS Nano, the device is able to “retain the same robust standards of benchtop lab-based tests.”
Go Anywhere Technology Improves POC Testing
Nucleic acid detecting assays are crucial tools anatomic pathologists use to identify pathogens, detect residual disease markers, and identify treatable mutations of diseases. Due to the need for amplification of nucleic acids for detection with benchtop equipment, there are challenges associated with providing rapid diagnostics outside the clinical laboratory.
Using the new mobile POC nucleic acid testing system developed by Ozcan et al, pathologists can effectively step away from the lab to perform rapid POC testing and accelerated diagnostics onsite, rather than needing to transport materials to and from a central laboratory. The mobile testing assay enables pathologists to carry a medical laboratory with them into the field, or into limited-resource or decentralized testing environments, without sacrificing quality or sensitivity. And according to the ACS Nano article, at a relatively low-cost compared to benchtop nucleic acid testing equipment.
The device developed by Ozcan Labs (above) is a “field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform [that] is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance. It is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.” (Caption and photo copyright: American Chemical Society.)
In an article published in Future Medicine, Ozcan and Hatice Ceylan Koydemir, PhD, a post-doctoral researcher in electrical engineering at UCLA, comment on the growing interest in mobile POC diagnostics, stating that smartphone-based devices and platforms have the potential “to be used for early detection and prevention of a variety of health problems.”
According to the article, smartphone-based sensing and imaging platforms have been developed to:
- Analyze chemicals and biological specimens;
- Perform advanced cytometry and bright-field/fluorescence microscopy;
- Detect bacterial contamination;
- Image nano-sized specimens;
- Detect antimicrobial drug resistance; and
- Analyze enzyme-linked immunosorbent assay (ELISA)-based testing.
Smartphones, according to Ozcan and Koydemir, have been adapted to a range of biomedical measurement tools, “have the potential to transform traditional uses of imaging, sensing, and diagnostic systems, especially for point-of-care applications and field settings,” and can provide speedy results.
A ‘Highly Stable’ and Sensitive System
The proof-of-concept study of Ozcan Lab’s new smartphone-based detection system and new detector dye mixture was led by Janay E. Kong, PhD in bioengineering at UCLA, with the help of Ozcan and fellow professors Dino Di Carlo, PhD, professor of bioengineering and mechanical and aerospace engineering at UCLA, and Omai Garner, PhD, associate professor of clinical microbiology at the David Geffen School of Medicine at UCLA.
According to an article in Bioscience Technologies, the new smartphone DNA detection system addresses issues with detection of light emitted from intercalator dyes, which are normally “too subtle and unstable for regular cellphone camera sensors.” The new system uses loop-mediated isothermal amplification (LAMP) to amplify DNA in connection with a newly developed dye that uses hydroxynaphthol blue (HNB) as an indicator.
The inclusion of HNB into the dye, according to the original research study, “yields 20 times higher fluorescent signal change over background compared to current intercalating dyes,” making the results bright enough for smartphone camera sensors without “interfering with the nucleic acid amplification process.” The original study reports that the digital LAMP system and use of the HNB intercalating dye, in fact, provided “significantly enhanced performance compared to a benchtop reader with standard LAMP conditions.”
Ozcan labs shows no signs of slowing down their development of mobile POC diagnostic devices. The development of these smartphone-based tools may provide unique and much-needed equipment for clinical pathologists given the rising interest in mobile healthcare worldwide.
— Amanda Warren
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UCLA Researchers Develop Lens-Free Smartphone Microscope, Pathologists May Be Able to Take the Clinical Pathology Laboratory Just About Anywhere
Smartphone “Dongle” Achieves Capabilities of Big Clinical Laboratory Analyzers: Diagnoses Three Diseases at Once from Single Drop of Blood
New Fast, Inexpensive, Mobile Device Accurately Identifies Healthcare-Acquired Infections and Communicates Findings to Doctors’ Smartphones and Portable Computers
Pathologists and Researchers Predict Development Trajectory for Biomarker-based Molecular Diagnostics in Support of Translational Medicine
Tiny, Simple-to-Use Lensless Microscope Might Soon Find a Place in Pathology
A legal, supervised injection site (SIS) affiliated with Vancouver Coastal Health found 86% of drugs tested with strips contained fentanyl when tested with these medical lab test kits
Here’s an unexpected application of point-of-care testing (POCT) that may surprise pathologists and medical laboratory leaders. In a sort of “guerilla-warfare” street experiment that applies diagnostic technologies to a problem, the manager of a needle-exchange program in the Bronx has been helping heroin and other opioid users discover if a product they are about to ingest is contaminated by handing out test strips designed for testing urine.
The addicts participating in these special programs use the POCT urine test strips to test their drugs for the presence of fentanyl, a powerful synthetic opioid analgesic similar to morphine that can increase the potency of opioids to lethal levels. Rehab program directors adopted this approach to help prevent overdoses and deaths among drug users.
Reducing Overdoses with Test Strip Handouts
Opioids such as morphine are often prescribed to cancer or surgery patients to treat severe pain. However, according to a National Institute on Drug Abuse (NIDA) fact sheet, fentanyl is “50 to 100 times more potent than morphine.” When fentanyl is mixed with heroin or cocaine and sold on the streets, the potent mix can be deadly, NIDA explained.
Test strips ordered from Canada designed to check patients’ urine for fentanyl are being used by St. Ann’s Corner of Harm Reduction (St. Ann’s) in the Bronx, New York. The strips are being used to check drug users’ syringes for fentanyl, according to a National Public Radio Shots article. The idea is to inform drug users of what they have in hand and possibly encourage them to choose not to take the drug, use less, or slow things down, Shots reported.
“If you’re doing dope, we’ll give you a test strip so you can test and see if there’s fentanyl,” stated Van Asher, Data Manager at St. Ann’s, in the Shots article.
Whether an unlicensed individual distributing test strips to drug users violates state or federal regulations was not broached in the Shots article.
St. Ann’s gives out about 15 strips a day each costing $1, Shots noted. St. Ann’s staff is sharing data collected on the encounters with the Centers for Disease Control and Prevention (CDC) and with New York health departments.
Finding Fentanyl with Test Strips in Canada
St. Ann’s isn’t the first to use urine test strips for drug checking. Vancouver Coastal Health (VCH) in British Columbia, Canada, launched a pilot program for drug-checking in 2016 at its Insite facility.
Insite is a supervised injection site (SIS). It opened its doors in 2003 and operates under a constitutional exemption to Canada’s Controlled Drugs and Substances Act.
At Insite’s “supervised injection site” facility (above) in Vancouver, British Columbia, drug users can “legally” inject illegal drugs. Directors of this program have adapted point-of-care urine test kits typically used in medical laboratory testing to allow drug users to test their heroin and opiate drugs for the presence of fentanyl. The goal is to reduce overdoses and deaths from users unknowingly ingesting fentanyl. (Photo copyright: CBCNews.)
Insite began to test drugs for the presence of fentanyl in the fall of 2016. Data from 173 tests performed in July and August found that 86% of drugs tested contained fentanyl, noted a VCH news release.
“These initial results confirm our suspicion that the local drug supply is overwhelmingly contaminated with fentanyl. We’re hoping this information can help people who use drugs,” stated Mark Lysyshyn, MD, MPH, VCH Medical Health Officer and Professor of Medicine at University of British Columbia.
The test works when the client dilutes the substance with a few drops of water. A positive or negative result for fentanyl is revealed within seconds.
The test strip used by Insite was designed to check for fentanyl in urine, not for checking drugs, noted the VCH statement. Insite intends to review the pilot program test data and decide whether to continue testing services after the pilot program concludes.
Alexander Walley, MD, Director of the Addiction Medicine Fellowship Program and Assistant Professor of Medicine at Boston Medical Center, stated the test may aid users’ decision-making.
“Even when they know they’re going to be positive for fentanyl, the experience of somebody testing their drugs and seeing that it’s fentanyl has an impact. It really encourages them to use more safely,” he stated in the Shots article.
Overdose Deaths Due to Fentanyl in America
A CBC News, Manitoba, article called the death rate due to fentanyl ingestion a “Canada-wide disaster.” However, the problem is significant in the US as well.
Death rates from synthetic opioids, including fentanyl, rose more than 72% from 2014 to 2015 in the US, according to the CDC.
In New York City, fentanyl is increasingly being linked to overdoses. In 2016, nearly half (44%) of drug deaths involved drugs mixed with fentanyl. That’s a 16% increase over 2015, according to a NYC Health press release.
A report from the Tennessee Department of Health noted that 1,451 people died from drug overdose in 2015. That’s a state record. Deaths associated with fentanyl rose significantly from 69 in 2014 to 174 in 2015, the report noted.
How Fentanyl Works and Why It Is Dangerous
Here are some fentanyl facts from the NIDA:
- Fentanyl works by binding to opioid receptors located in areas of the brain that control pain and emotions;
- People may experience side effects such as euphoria, drowsiness, nausea, confusion, addiction, respiratory arrest, unconsciousness, coma, and death;
- Increased risk of overdose exists when drug users are unaware a drug they are ingesting contains fentanyl.
Clinical laboratory directors and pathology groups nationwide might want to follow the progress of test strip services at St. Ann’s Corner and Insite’s SIS. This twist on traditional POCT—using urine test strips to look for the presence of fentanyl in substances—could aid their own communities achieve public awareness, change behaviors, and save lives.
—Donna Marie Pocius
An Experiment Helps Heroin Users Test Their Street Drugs for Fentanyl
86% of Drugs Checked at Insite Contain Fentanyl
Fentanyl Deaths Are a Canada-wide Disaster
5 New Supervised Injections Sites Coming to Fight Vancouver’s Fentanyl Overdose Crisis
1,451 Tennesseans Die from Drug Overdoses in 2015
State Data Confirms Overdose Deaths Primarily White Opioid Users
New low-cost alternatives to emergency department and hospital visits could require flexibility from pathology groups and clinical laboratories to provide the best quality care
In response to the rising cost of conventional hospital services, innovative healthcare models such as micro-hospitals, bedless hospitals, and mobile and freestanding emergency rooms (ERs), are attempting to lower costs while maintaining quality of care by providing alternatives to traditional ER visits and hospital stays.
This means new challenges and opportunities for pathology groups and medical laboratories that can adapt to the different needs of these new healthcare delivery models. Each different care model will want clinical lab testing services and the reporting of lab test results to be handled in ways that enable these providers to achieve improved patient outcomes. (more…)
As cognitive and cloud computing continue to advance, and mobile technologies become more accessible across the globe, innovative apps and mobile attachments are using algorithms to replace the need for complex and time-consuming diagnostic tests
Mobile healthcare—also known as mHealth—is attracting plenty of research dollars as entrepreneurs look for ways improve consumers’ access to various medical services in ways that could reduce healthcare costs. For that reason, some mHealth solutions may be used by clinical laboratories and pathology groups to give patients faster access to diagnostic services and information about medical laboratory tests.
Most mHealth solutions excel at doing a single, defined task well. In some cases, they are faster and as accurate as human-based testing or observation. However, few solutions can tackle complex diagnostics, such as determining the pathogens involved in sepsis. And mHealth cannot replace the human element of communication and empathy, which will always have a place in the medical process. (more…)