A novel tool that detects the presence of specific nucleic acid sequences is the basis for a new family of point-of-care (POC) diagnostics, which could take the form of an at-home test strip in the next few years.
CRISPR (clustered regularly interspaced short palindromic repeats) represents a bacterial immune system that contains a programmable protein able to cut DNA or RNA. With this technology, genes can be edited and modified in living cells and organisms. While much effort has gone into developing CRISPR to treat disease, CRISPR is also a platform for diagnostics.
CRISPR proteins are able to generate a signal in real-time when they match up with DNA or RNA sequences. In this way, CRISPR can be programmed to detect the presence of specific nucleic acid sequences.
This aspect of CRISPR is driving the development of new molecular diagnostics. One CRISPR protein, Cas14, which contains up to 700 amino acids, can detect double-stranded and single-stranded DNA, as well as RNA, and is able to detect a bacteria or virus.
DNA and RNA sensing
Scientists at Mammoth Biosciences in South San Francisco, CA, are using CRISPR to build a disease detection platform capable of targeting any biomarker or disease with DNA or RNA. Last year, the company received an exclusive license from the University of California, Berkeley to Cas14, and it intends to develop an easy and affordable POC test. The company also was issued two U.S. patents (Nos. 10,253,365 and 10,337,051) that cover comprehensive techniques enabling Mammoth to offer DNA and RNA detection as CRISPR diagnostics.
"Similar to the way Google built a search engine for the web, Mammoth Biosciences has used CRISPR to build the search engine for biology," Trevor Martin, PhD, CEO and co-founder of Mammoth Biosciences, explained in an email. "This search engine can be used to find and edit cells for therapeutic uses as is traditionally done, or Mammoth's CRISPR-based platform can also search and find nucleic acids that are indicative of disease in samples ranging from blood to saliva, and report these results back as a diagnostic."
According to Martin, the company's CRISPR-based detection platform can sense any biomarker or disease by detecting DNA or RNA.
"The base functionality of a CRISPR system is created by 'programming' a Cas enzyme to bind to a certain DNA or RNA sequence by designing a guide RNA that is complementary to the sequence you want the enzyme to interact with," he said.
For editing, it's possible to design a guide RNA to target the Cas protein to a gene to be edited. When the protein finds this sequence, the built-in molecular "scissors" of the Cas protein can cut the nucleic acid at that location, which can then be leveraged to create an edit.
Finding the sequence
"For diagnostics, we leverage this same 'programming' of the Cas enzyme through a guide RNA, but instead of sending the protein to a gene we want to edit, we send the protein to find a DNA or RNA sequence that is unique to what we want to detect -- for example, an RNA or DNA molecule that specifically indicates a disease," Martin continued.
"To leverage this binding for diagnostics, we have developed special classes of enzymes that, if they find their target DNA or RNA sequence, actually cut many orders of magnitude more DNA or RNA more broadly in the sample," he added.
The company leverages this "molecular switch" activity by including a reporter molecule in the reaction that, for example, releases color when it's cut. In this way, a single detected DNA or RNA sequence results in a color change reaction that can be used to indicate if the target is present or absent, Martin explained.
Mammoth Biosciences intends to create a POC test that's as simple as adding a liquid sample to a disposable device and diagnosing the result through a Mammoth app within 30 minutes, he said.
"As an example, you could potentially detect a disease like malaria in saliva by having the patient spit in a tube, and then adding the CRISPR Cas protein, the guide RNA specific to malaria, and the reporter molecule to the tube as well, then reading out the presence or absence of malaria through a color change of the tube," he said.
Martin indicated that the Mammoth test does not require refrigeration or fixed instrumentation, so doctors can go straight to the point of care and diagnose in real-time.
The test could be leveraged in all settings -- from the hospital, to the point of care, to the home. The speed and accuracy of the test make it a particularly interesting technology for POC and home use cases, Martin explained. The test could take the form of disposable strips or a highly multiplexed device. The first test could be available in the next few years, he added.
CRISPR and synthetic biology
Meanwhile, another startup that is also applying CRISPR to diagnostics launched in March 2019. Sherlock Biosciences in Cambridge, MA, is using CRISPR and synthetic biology, which involves engineering biological systems to have new capabilities, to create novel molecular diagnostics.
"There is a need for new diagnostic tools that will have a great impact on healthcare," said Rahul Dhanda, Sherlock's co-founder, president, and CEO, in an interview. "CRISPR technology provides an ability to develop a very accurate and very rapid diagnostic."
Sherlock Biosciences was named after one of its foundational platform technologies -- specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) -- which it exclusively licensed from the Eli and Edythe L. Broad Institute, a biomedical and genomic research center that is a part of both the Massachusetts Institute of Technology and Harvard University.
SHERLOCK was developed by a team led by Feng Zhang, PhD, company co-founder and chair of Sherlock's scientific advisory board, who collaborated with co-founder Jim Collins, PhD. It was created as a way to identify specific genetic targets using CRISPR, and SHERLOCK can detect the unique genetic fingerprints of virtually any DNA or RNA sequence in any organism or pathogen, according to the company.
Signaling molecules
The SHERLOCK platform amplifies genetic sequences and programs a CRISPR molecule to detect the presence of a specific genetic signature in a sample, which can also be quantified. When it finds those signatures, the CRISPR enzyme activates signaling molecules for detection. This signal can be adapted to work on a simple paper-strip test or on laboratory equipment, or it could provide an electrochemical readout that can be read with a mobile phone, explained Dhanda.
The technology has shown improved performance when used on clinical specimens, he said.
"The key advantages of the technology are its speed to results and the fact that it is highly accurate," Dhanda continued. "It's a very active process, with time from sample analysis to results in 10 minutes or less. The speed to getting test results makes the technology viable for POC use where immediate results are critical, eliminating the need for a patient to return to a clinic to obtain test results at a later date."
"SHERLOCK is a versatile platform that is easy to use on any device, including portable handheld devices, lending the technology to POC use," he said.
He indicated that Sherlock Biosciences is initially targeting CRISPR diagnostics to infectious diseases. The technology is approximately two years from clinical trials for POC targets and three years from being a commercial test, which would be available first in the U.S. and eventually internationally.
