In a new study published in Science, investigators adapted an RNA-targeting CRISPR enzyme as a rapid, inexpensive, and highly sensitive diagnostic tool.
 
The novel tool, named SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), has the potential to transform the research landscape and global public health by detecting cancer, monitoring antibiotic resistance, and responding to viral and bacterial outbreaks.
 
“It’s exciting that the Cas13a enzyme, which was originally identified in our collaboration with Eugene Koonin to study the basic biology of bacterial immunity, can be harnessed to achieve such extraordinary sensitivity, which will be powerful for both science and clinical medicine,” said investigator Feng Zhang.
 
The RNA-targeting CRISPR enzyme Cas13a, previously known as C2c2, was first characterized by Zhang and his colleagues in June 2016. Unlike DNA-targeting CRISPR enzymes, Cas13a can stay active after splicing its RNA target, and may even continue to cut other non-targeted RNAs in a burst of activity the investigators refer to as “collateral cleavage.”
 
Prior studies have harnessed Cas13a collateral cleavage activity for RNA detection, but it requires millions of molecules, therefore it lacked the sensitivity needed.
 
The new method, however, is substantially more sensitive, and is the result of a collaborative effort surrounding diagnostics for the Zika virus. In 2014, the investigators created a synthetic paper-based test for the Ebola virus that could be both shipped and stored at room temperature.
 
After investigators modified the system later to detect the Zika virus, they demonstrated how applying low-levels of applied heat to the samples boosts the amount of RNA.
 
The investigators used a different amplification process that instead relied on body heat to boost the levels of DNA or RNA in the test samples. Once the levels increased, a second amplification step was added to convert the DNA to RNA. This allowed the investigators to increase the sensitivity of the RNA-targeting CRISPR by a million-fold.
 
“We can now effectively and readily make sensors for any nucleic acid, which is incredibly powerful when you think of diagnostics and research applications,” said investigator Jim Collins. “This tool offers the sensitivity that could detect an extremely small amount of cancer DNA in a patient’s blood sample, for example, which would help researchers understand how cancer mutates over time. For public health, it could help researchers monitor the frequency of antibiotic-resistant bacteria in a population. The scientific possibilities get very exciting very quickly.”
 
The scientists demonstrated the versatility of the method on a range of applications, including: detecting the presence of Zika in patient blood or urine samples within hours; distinguishing between the genetic sequences of African and American strains of Zika virus; discriminating specific types of bacteria, such as E. coli; detecting antibiotic resistance genes; identifying cancerous mutations in simulated cell-free DNA fragments; and rapidly reading human genetic information, such as risk of heart disease, from a saliva sample.
 
The most obvious use for the novel diagnostic tool would be as a rapid, point-of-care diagnostic for infectious disease outbreaks in areas with limited resources.
 
“There is a great excitement around this system,” said co-author Deb Hung. “There is still much work to be done, but if SHERLOCK can be developed to its full potential it could fundamentally change the diagnosis of common and emerging infectious diseases.”
 
A main benefit of the tool is that it can be used as a paper-based test that would not require refrigeration, meaning it could be used for fast deployment and widespread use outside of traditional settings.
 
“One thing that’s especially powerful about SHERLOCK is its ability to start testing without a lot of complicated and time-consuming upstream experimental work,” said co-author Pardis Sabeti. “This ability to take raw samples and immediately start processing could transform the diagnosis of Zika and the boundless number of other infectious diseases. This is just the beginning.”