The CRISPR Diagnostic Toolkit Gets Handier

The CRISPR Diagnostic Toolkit Gets Handier

In December, we reported the emergence of a CRISPR-based tool known as SHERLOCK, whose rapidity and extreme sensitivity defined its promise in the realm of diagnostics. In February, a few new studies related to the gene editing technique were documented inScience, one of which is a new diagnostic application, and the other an update on still-nascent SHERLOCK. For more information on CRISPR, see Kalorama’s white paper on gene editing and full report on the technique’s markets.

To review, SHERLOCK—Specific High-sensitivity Enzymatic Reporter unlocking—is derived from CRISPR/Cas; like its progenitor, the technique employs guide RNAs paired up with enzymes, seeking out specific DNA sequences and cutting them. However, the Cas13a enzyme used in SHERLOCK (as opposed to CRISPR’s Cas9) makes additional cuts near the target cleavage site, as well as to an additional reporter RNA molecule that releases a fluorescent signal when cleaved. Combined with SHERLOCK’s isothermal amplification and its high sensitivity, achieved through multiple amplification steps, the method showed vast potential asin vitrodiagnostic technology.

SHERLOCK is back in the news with an update. As reported in an article published by the AAAS on 15 February,1SHERLOCKv2 shows a 3.5-fold increase in sensitivity over its original iteration; responsible for this is the use of Csm6, an auxiliary CRISPR-associated enzyme, in addition to Cas13. It was found that not only does this further amplify the signal, but it provides for more rapid detection by lateral flow, making the technique even more viable for applications in paper-based diagnostic tests without the need for expensive, complex machinery. Furthermore, researchers, including original CRISPR developer Jennifer Doudna, were able to use SHERLOCK for multiplex detection, reporting that they designed a test able to simultaneously find Zika and Dengue virus ssRNA as well as a synthetic ssRNA target. With preamplification by recombinase polymerase amplification, single-molecule detection can be achieved at the attomolar level.

Described in another article published on the same day2is an altogether new platform derived from CRISPR/Cas. A team led by another CRISPR pioneer, Feng Zhang, reports that the enzyme Cas12a, after binding to and cleaving the target DNA sequence, will continue to cleave and ultimately completely degrade surrounding ssDNA molecules. Combining this ssDNase activation with isothermal amplification, Zhang and crew developed a technique they call DNA Endonuclease Targeted CRISPRTransReporter, or DETECTR, which, similarly to SHERLOCK, can achieve attomolar sensitivity for DNA detection. In the study, DETECTR wasused to specifically detect human papillomavirus strains 16 and 18 from cultured human cells (SiHa, HeLa, and uninfected BJAB as control) as well as from patient anal swab samples within one hour, as a proof-of-concept for a new molecular diagnostic platform. However, author Janice Chen said that the enzyme’s cut-without-prejudice operation may have unwanted effects, including the possibility of Cas12a cutting single stranded DNA created by the cell as a normal product of gene transcription. As always, caution is advised and further research is necessary.

Finally, in unrelated-but-still-somewhat-related news: In the previous blog entry on CRISPR, we briefly mentioned that potential issues could emerge in the form of mosaics produced by the guide RNA missing target sequences, resulting in widespread damage to the genome. However, this week,Nature Methodshas retracted an article published in May 2017 outlining the danger.3Initially, the article sparked an outcry from a number of people, some of whom were associated with companies with licensing rights to CRISPR; criticism included assertions that the investigators did not use proper controls to ensure that mutations were not results from normal variation in subjects.Nature Methodsitself published an editorial statement of concern in July 2017,4and issued the formal retraction in late March.5The authors, led by Stanford’s Vinit B Mahajan, acknowledged that the criticism may be on point;6Mahajan and three coauthors nonetheless disapproved of the retraction, however, stating that the matter requires further study before such a statement can be made in full faith. Follow-up studies are underway using whole-genome sequencing.

Sources:

1 Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. (2018) Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science; posted online 15 February 2018, accessed 03 April 2018. DOI: 10.1126/science.aaq0179

2 Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity.Science; posted online 15 February 2018, accessed 03 April 2018. DOI: 10.1126/science.aar6245

3 Schaefer KA, Wu W-H, Colgan DF, Tsang SH, Bassuk AG, Mahajan VB. (2017) Unexpected mutations after CRISPR-Cas9 editingin vivo. Nature Methods; 14(6), 547-8. DOI: 10.1038/nmeth.4293

4  http://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293.html#correction2

Published 25 July 2017, accessed 04 April 2018

5  http://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293.html#correction3

Published 30 March 2018, accessed 04 April 2018

6 Schaefer KA, Darbro BW, Colgan DF, Tsang SH, Bassuk AG, Mahajan VB. (2018) Corrigendum and follow-up: Whole genome sequencing of multiple CRISPR-edited mouse lines suggest no excess mutations. bio℞iv154450 (preprint); published online 26 March 2018. DOI: 10.1101/154450