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Crispr-Based Diagnostics System Designed

Crispr-Cas9 illustration

Crispr-Cas9 illustration (LBL.gov)

14 April 2017. A bioengineering team from Harvard University and Massachusetts Institute of Technology designed simple, inexpensive diagnostics tools to detect infectious diseases, based on Crispr, an emerging genome-editing technology. A report of the technology appears in this week’s issue of the journal Science (paid subscription required).

Researchers from the Broad Institute, a medical research center affiliated with Harvard and MIT, as well as labs from the two institutions, are seeking to make detection of disease simpler and less costly for clinicians, particularly those in low-resource regions of the world. The team led by Broad Institute bioengineer Feng Zhang, also on the faculty at MIT, applied Crispr — short for clustered regularly interspaced short palindromic repeats — to the task. Zhang is among the pioneers in Crispr technology.

Crispr is based on bacterial defense mechanisms that use RNA to identify and monitor precise locations in DNA. The actual editing of genomes with Crispr employs enzymes that cleave DNA strands at the desired points, with Crispr-associated protein 9, or Cas9, being the enzyme used for the longest period. In this project, Zhang and colleagues, focus Crispr on RNA, nucleic acids carrying instructions to cells from the genetic code in DNA. The team also harnesses a different enzyme known as Cas13a, with properties that turn Crispr into a tool for disease detection.

Cas13a targets RNA rather than DNA, but also exhibits what the researchers call promiscuous behavior, in that it keeps cleaving RNA strands after the first cuts. The researchers adapted earlier work with this enzyme, then code-named C2c2, and increased its sensitivity, so it could work with tiny specimen samples, in some cases as small as a single molecule. To develop this property of Cas13a for diagnostics, Zhang partnered with James Collins, one of the core faculty at Harvard’s Wyss Institute, a bioengineering research center.

As reported in Science & Enterprise, Collins and colleagues in 2014 developed a simple, paper-based diagnostics system to quickly detect complex cellular reactions from small specimen samples, including those for the Ebola virus, which at the time was ravaging West Africa. Zhang and Collins applied many of those same principles to Crispr diagnostics with processes, such as low heat, to amplify DNA in specimen samples. The team also employed a technique called recombinase polymerase amplification to convert amplified DNA to RNA for detection by Cas13a. These methods enable Cas13a to emit signals about the RNA being cleaved for measurement and assessment.

The team call its technology platform Specific High Sensitivity Enzymatic Reporter Unlocking, or Sherlock, which the researchers tested with a number of different infectious disease samples. The tests show Sherlock can discriminate between small samples of Zika and dengue viruses, even with the amplification reagents freeze-dried and reconstituted with water later on, similar to processes used in remote clinics. Further tests show Sherlock able to detect Zika viruses in tiny blood, urine, and saliva samples, as well as potentially predict viral loads from Zika in patients.

In addition, Sherlock can distinguish between different bacteria and viral strains. The researchers report Sherlock analyzes DNA to detect dangerous bacteria to humans, such as E. coli and Pseudomonas aeruginosa, associated with health care facility infections, but also can discriminate among various strains of those bacteria. The technology likewise distinguishes between African and American Zika strains, and different dengue viruses.

Moreover, the researchers show Sherlock can analyze DNA samples for human genetic characteristics. The team collected saliva samples from 5 individuals, and used Sherlock to identify common genetic variations known as single-nucleotide polymorphisms, or SNPs. The researchers then compared Sherlock’s analyses with reports from the personal genetics company 23andMe, and found within 5 minutes that they matched. Further tests also show Sherlock can detect 2 cancer-causing mutations in blood samples.

The authors believe Sherlock has wide applications in diagnostics, particularly in simple paper-based tests that can be produced for as little as $0.61 each. The universities already applied for patents on the technologies. The web site of Editas Medicine, the company founded by Zhang and others to commercialize Crispr technology, so far lists no diagnostics in its product pipeline.

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