Saturday, January 19, 2019

Genetic Spying for diagnostics- SHERLOCK CRISPR System: An update

Greetings

There have been so many great microbiology stories that I should have written about in the year 2018, but I was so occupied I never got the time. Well, doesn't mean this blog has died. Of course, the "almost a year gap" in writing has slightly affected my style of blogging. But I will pick it up back in a few weeks. That being said, let us come back to talk some science.

In the field of diagnostics, speed and accuracy of diagnostics is an ever challenging factor. From classic microbiology techniques which takes at least 48 hours to identify and characterise a pathogen, we have come a long way to more modern molecular methods such as PCR which can report in a couple of hours. These days, bacterial diagnostics have become faster with the use of MALDI-TOF instrumentation. For a quick and reliable diagnostics we still mostly rely on PCR and NGS methods. But they come with a high price tag and sophistication which are not directly "Field deployable". An alternative is immunologically based rapid diagnostic tests which take months to develop and validate and have lower capabilities than a genetic test.

On a side note, the CRISPR-Cas system has been widely harvested in genetic engineering. There is a great debate on who owns the intellectual property rights to CRISPR based gene editing technology which is an ongoing legal battle between the Broad and Berkley institute. There is also news of a Chinese research team lead by He Jiankui claiming to have created the first CRISPR-edited twins girls named Lulu and Nana. However, there are strong doubts over the claim and the bioethical aspects of this work have been subject an international discussion (Link).

Cas 13 protein has some interesting properties. Cas13a is functionally comparable to Cas9. In addition to its programmable RNase activity, Cas13a shows a collateral activity leading to non-specific degradation of any nearby transcripts regardless of complementarity to the spacer. Upon recognition of a specific RNA sequence programmed Cas13a is activated which then cleaves the surrounding ssRNA molecules. By using a quenched fluorescent ssRNA which can be cleaved to release the reporter (The idea is similar to TaqMan chemistry in Real-time PCR), the signal can be easily read. This phenomenon was harnessed by a group of scientists from the Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, the Institute for Medical Engineering & Science at MIT, and the Wyss Institute for Biologically Inspired Engineering at Harvard University to develop a technique called as "SHERLOCK" in April 2017. SHERLOCK is a fancy acronym for Specific High-sensitivity Enzymatic Reporter unLOCKing. Subsequently the same team in April 2018 reported an improved design further to develop a protocol known as SHERLOCK-HUDSON. HUDSON is an acronym for "Heating Unextracted Diagnostic Samples to Obliterate Nucleases). Originally SHERLOCK presented with a few limitations related to quantification. Subsequently, SHERLOCK V2 which uses a combination of Cas13, Cas12a, and Csm6, was reported which can achieve multiplex nucleic acid detection with enhanced sensitivity. Also, SHERLOCK used fluorescent detectors whereas, in the improved version the reporter was modified such that cleaved reporter could be detected on commercial lateral flow strips.

There are a couple of associated technologies here. HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) platform for nucleic acid detection uses Cas 12 as the enzyme. DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter which was independently developed at Doudna's Lab also uses Cas 12a with small changes in the details.

Fig 1: Overview of the development of the CRISPR systems and their applications. Source

Fig 2: The principle of SHERLOCK and DETECTR detection methods.
SHERLOCK is mainly an RNA detection tool and has been demonstrated to be useful in diagnostics such as Zika Virus detection at attomolar concentrations. In contrast, DETECTR is a DNA detection method and has been demonstrated to be useful in identifying HPV. HUDSON is not a detection method, but rather a processing protocol where the virus particles are lysed and heat treated to work directly with SHERLOCK without the need for separate processing of samples. HOLMES works very much similar to other techniques except that it uses Cas12b in a Loop-Mediated Isothermal Amplification methodology. Figure 2 gives a schematic summary of the working of SHERLOCK and DETECTR.

Feng Zhang, whose lab is involved in developing the SHERLOCK technology commented, "SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method for detecting nucleic acid material — and that can mean a virus, tumour DNA, and many other targets. The SHERLOCK improvements now give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications. The technology demonstrates potential for many healthcare applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer, but it can also be used for industrial and agricultural applications where monitoring steps along the supply chain can reduce waste and improve safety".

References:

Liu H, Wang L, Luo Y. Blossom of CRISPR technologies and applications in disease treatment. Synth Syst Biotechnol. 2018 Oct 22;3(4):217-228. Link

Myhrvold et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018 Apr 27;360(6387):444-448. Link

Gootenberg et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017 Apr 28;356(6336):438-442. Link