Written by Gouri Ajith and Edited by Emily Majorkiewicz
In regions of the world affected by war, natural disaster, or extreme poverty, limited access to medicine and health services leaves people extremely vulnerable to disease. Without trained personnel and expensive equipment, it is often difficult to detect infectious diseases before patients become seriously ill. But, with the recent development of the simple diagnostic tool SHERLOCK, making disease detection accessible in these situations is suddenly feasible. These tools utilize the gene-editing technology CRISPR to detect characteristic viruses and bacteria in human samples. CRISPR, which stands for “clusters of regularly interspaced short palindromic repeats,” uses crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as a guide to target a specific DNA sequence and an enzyme called Cas to cut the DNA at specific, predetermined sites [1]. Scientists can then insert a desired sequence into these cut sites, correcting disease-causing genetic mutations or activating the expression of certain genes [2]. Recent studies conducted by the lab of Feng Zhang have been able to utilize CRISPR to do more than edit genomes; CRISPR can now effectively detect deadly infectious diseases.
This new detection tool SHERLOCK, which stands for “specific high sensitivity enzymatic reporter unlocking,” uses the Cas enzyme of the CRISPR system to target and cut the single-stranded genetic molecule RNA, instead of DNA [3]. Once the Cas enzyme has cut the target viral RNA, it begins cutting any nearby RNA that is also present [3]. Scientists have utilized this behavior to induce fluorescent signals that reveal if the viruses or bacteria characteristic of certain diseases are present. Fluorescent RNA is added to the samples that are being tested to act as a reporter, and once the Cas enzyme has cut the viral RNA, it snips the nearby fluorescent RNA and causes fluorescence, indicating the presence of the virus [3]. Zhang’s lab was able to use SHERLOCK to target the RNA of Zika, dengue, and other viruses and bacteria in human samples [3]. A second, more recent study conducted by the same lab has shown how SHERLOCK can be improved to simultaneously detect multiple viruses and bacteria in the same sample [4]. This is achieved by introducing multiple Cas enzymes that work together to cut various viral and bacterial RNA sequences [4]. The effectiveness of SHERLOCK is in part due to its high sensitivity; it can detect even trace levels of virus or bacteria in a sample [5].
In addition to detecting viral and bacterial RNA, SHERLOCK can also target DNA sequences, by first using an enzyme to transcribe the DNA in the sample to RNA [3]. The Cas enzymes can then target and signal the presence of low-frequency cancer mutations and single nucleotide polymorphisms, variations in the DNA sequence associated with certain diseases [3]. Not only is SHERLOCK more sensitive than the currently used diagnostic tools, which target proteins, it is also quicker and more economical [5]. In addition to fluorescent signals, Zhang’s lab has also developed a version of SHERLOCK that uses simple fiber paper strips to detect the target RNA; like a pregnancy test, the paper is dipped into the sample and a dark line appears if the virus or bacteria is present [3]. As it costs only 61 cents and does not require complex machinery or electricity, the tool could be easily utilized in war-torn or poverty-stricken areas [3]. If SHERLOCK’s method of viral and bacterial DNA detection is approved by government regulatory agencies and is commercially mass produced, it could make disease diagnosis globally accessible.
References
[1]Jeffry D. Sander, J. Keith Joung. 2014. CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology. 32: 347–355.
[2] “Questions and Answers About CRISPR.” Broad Institute, https://www.broadinstitute.org/what-broad/areas-focus/project-spotlight/questions-and-answers-about-crispr.
[3]Jonathan S. Gootenberg, Omar O. Abudayyeh, Jeong Wook Lee, Patrick Essletzbichler, Aaron J. Dy, Julia Joung,Vanessa Verdine, Nina Donghia, Nichole M. Daringer, Catherine A. Freije, Cameron Myhrvold, Roby P. Bhattacharyya, Jonathan Livny, Aviv Regev, Eugene V. Koonin, Deborah T. Hung1, Pardis C. Sabeti, James J. Collins, Feng Zhang. 2018. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 356: 438-442.
[4]Jonathan S. Gootenberg, Omar O. Abudayyeh, Max J. Kellner1, Julia Joung, James J. Collins, Feng Zhang. 2018. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 360: 439-444.
[5] “New CRISPR tool can detect tiny amounts of viruses.” Science, http://www.sciencemag.org/news/2017/04/new-crispr-tool-can-detect-tiny-amounts-viruses.