Friday, April 27, 2018

CRISPR Used for Infectious Disease Diagnostics

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) are short, repeated DNA sequences found in the genomes of bacteria and other microorganisms. These sequences help to fight off bacteriophages (bacterial viruses) by slicing the invading viruses, thereby preventing the virus from replicating.

CRISPR is now being used for a variety of applications. For example, CRISPR can facilitate the replacement of a mutant gene with the correct sequence, thereby curing a genetic disorder. CRISPR is now being explored to modify antibiotics that are specific for pathogenic bacteria without impacting good bacteria that the body requires.

The Harvard Graduate School of Arts and Sciences has developed an easy to understand website for reviewing CRISPR. It includes an illustration of the basic principles of CRISPR, shown below (click for a larger image):

Science Magazine has recently published an article on how CRISPR can be used for infectious disease diagnostics. Initial areas of focus target Zika, Dengue and human papillomavirus.

Different diagnostic methods are being developed which exploit the power of CRISPR technology. For example, double stranded (ds) DNA may be extracted from a sample and amplified using isothermal preamplification by recombinase polymerase amplification (RPA). The target sDNA amplicons are then sequence specifically cleaved by a Cas12a-crRNA protein complex, which activates nonspecific cleavage of single stranded (ss) DNA. A fluorescent dye binds to the ssDNA to create a detection signal. This process is known as DNA endonuclease-targeted CRISPR trans reporter (DETECTR).

HUDSON (heating unextracted diagnostic samples to obliterate nucleases) is a method by which heat and chemicals inactivate ribonucleases (RNases) and lyses viral particles, thereby releasing nucleic acids into solution. This can be combined with SHERLOCK (specific high-sensitivity enzymatic reporter unlocking), a Cas13-based nucleic acid detection method, to deliver a rapid (1-2 hour) diagnostic test. Using fluorescent readouts, the test can give a positive-negative readout on a paper test-strip. Read more about each of these methods, which are summarized in the illustration below (click for a larger image) with accompanying references, in Science Magazine.


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