CRISPR-based diagnostic tool
Researchers have adapted an RNA-targeting CRISPR protein for use as “a rapid, inexpensive, highly sensitive diagnostic tool”.
The scientists demonstrate the method’s versatility on a range of applications, including: detecting the presence of Zika virus 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.
Because the tool can be designed for use as a paper-based test that does not require refrigeration, the researchers say it is well suited for fast deployment and widespread use inside and outside of traditional settings — such as at a field hospital during an outbreak, or a rural clinic with limited access to advanced equipment.
The findings by a team of scientists, from the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering and Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, have been published in Science.
In June 2016, MIT’s Feng Zhang and his colleagues first characterised the RNA-targeting CRISPR enzyme, now called Cas13a (previously known as C2c2), which can be programmed to cleave particular RNA sequences in bacterial cells. Unlike DNA-targeting CRISPR enzymes (such as Cas9 and Cpf1), Cas13a can remain active after cutting its intended RNA target and may continue to cut other nontargeted RNAs in a burst of activity that Zhang lab scientists referred to as “collateral cleavage”. In their paper and patent filing, the team described a wide range of biotechnological applications for the system, including harnessing RNA cleavage and collateral activity for basic research, diagnostics and therapeutics.
In a paper in Nature in September 2016, Jennifer Doudna, Alexandra East-Seletsky and their colleagues at the University of California at Berkeley employed the Cas13a collateral cleavage activity for RNA detection. That method required the presence of many millions of molecules, however, and therefore lacked the sensitivity required for many research and clinical applications.
The latest method is “a million-fold more sensitive”. This increase was the result of a collaboration between Zhang and his team and Broad Institute member Jim Collins, who had been working on diagnostics for Zika virus.
Working together, the Zhang and Collins teams were able to use a different amplification process, relying on body heat, to boost the levels of DNA or RNA in their test samples. Once the level was increased, the team applied a second amplification step to convert the DNA to RNA, which enabled them to increase the sensitivity of the RNA-targeting CRISPR by a million-fold, all with a tool that can be used in nearly any setting.
“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 Collins, the Termeer Professor of Medical Engineering and Science at MIT and core faculty member at the Wyss Institute. “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.”
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