jueves, 4 de mayo de 2017

Quick, sensitive diagnostic tests with CRISPR | National Institutes of Health (NIH)

Quick, sensitive diagnostic tests with CRISPR | National Institutes of Health (NIH)

National Institutes of Health (NIH) - Turning Discovery into Health

Quick, sensitive diagnostic tests with CRISPR

At a Glance

  • Scientists developed a CRISPR-based tool that can detect tiny amounts of Zika and Dengue virus, distinguish pathogenic bacteria, and identify DNA variations.
  • The components of the system can be freeze-dried and reconstituted on paper for field applications, opening the door to many practical uses.
Illustration of RNA moleculesThe new tool can detect and distinguish tiny amounts of genetic material.Svisio/iStock/Thinkstock
The ability to quickly and cheaply detect minute amounts of specific nucleic acid (DNA and RNA) sequences could bring significant public health benefits. For example, it could be used to detect viral or bacterial infections in a population during outbreaks. Other possible uses include finding antibiotic-resistance genes in bacteria or tumor mutations in the body. Current methods for detecting nucleic acids involve trade-offs in sensitivity, specificity, simplicity, cost, and speed.
Drs. James J. Collins and Feng Zhang of the Broad Institute of MIT and Harvard developed a new approach by adapting the CRISPR system, which bacteria use to defend themselves from other microbes. CRISPR enzymes use short “guide RNAs” to identify specific target sequences to cleave. Zhang’s group previously discovered that one CRISPR enzyme, called Cas13a, has an interesting “collateral effect.” After being activated by its target RNA sequence, Cas13a goes on to indiscriminately slice other non-targeted RNA nearby.
The researchers took advantage of this property to design a CRISPR-based nucleic acid detection platform. To detect when a target sequence was present, they added “reporter” RNA designed to emit a signal when cut. Whenever Cas13a was activated, it would go on to cut the reporter RNA and emit a signal. The study was funded in part by NIH’s National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Mental Health (NIMH). The team described their approach online in Science on April 13, 2017.
The scientists first tested Cas13a enzymes from different bacteria to identify which had the best RNA-guided cutting activity. As amounts of DNA and RNA in samples can be minute, the researchers applied a technique called recombinase polymerase amplification, which can amplify nucleic acids without special equipment. Another enzyme could also be added to the reaction to convert DNA to RNA for Cas13a detection.
The team called this system SHERLOCK. Tests showed that SHERLOCK could detect RNA or DNA molecules at minute levels called attomolar levels. Established nucleic acid detection approaches can be similarly sensitive, but SHERLOCK gave more consistent results.
The researchers demonstrated several potential uses. SHERLOCK was able to detect specific strains of Zika and Dengue virus. It could detect Zika virus in serum, urine, and saliva from patients. It could distinguish pathogenic strains of bacteria. It could distinguish single base differences in DNA extracted from human saliva. Finally, it could detect cancer mutations among DNA fragments at levels like that found in patient blood.
Notably, SHERLOCK yielded comparable results when its components were freeze-dried, reconstituted, and tested on glass fiber paper. The scientists calculated that a paper test could be designed and created in a matter of days for as little as $0.61 per test. These qualities highlight the potential of this system for diagnostic field applications.
“We can now effectively and readily make sensors for any nucleic acid, which is incredibly powerful when you think of diagnostics and research applications,” Collins says. “The scientific possibilities get very exciting very quickly.”
—by Harrison Wein, Ph.D.

Related Links

References: Nucleic acid detection with CRISPR-Cas13a/C2c2. Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F. Science. 2017 Apr 13. pii: eaam9321. doi: 10.1126/science.aam9321. [Epub ahead of print]. PMID: 28408723.
Funding: NIH’s National Institute of Allergy and Infectious Diseases (NIAID) and National Institute of Mental Health (NIMH); Paul and Daisy Soros Fellowship; National Defense Science and Engineering Fellowship; US Department of Energy; National Science Foundation; Air Force Office of Scientific Research; Marc and Lynne Benioff; Josh and Anita Bekenstein; Howard Hughes Medical Institute; New York Stem Cell Foundation; Simons Foundation; Paul G. Allen Family Foundation; Vallee Foundation; James and Patricia Poitras; Robert Metcalfe; and David Cheng.

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