DETECTing Merkel cell Polyomavirus in Merkel Tumours

Abstract

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post integration (~80% cases), or by UV mediated DNA damage. Interestingly, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this differential information of significant diagnostic and prognostic value. Also, MCV as a causative agent also provides a direct target for therapy in virus positive MCC patients. Currently, the methods used for diagnosis of MCV in tumours are often tedious, discordant and unreliable. In this study we used a guided molecular scissors based -DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme -gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC tumours, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that this sophisticated MCV DETECTR system can detect MCV integrated in Merkel tumour rapidly, specifically and with femto-molar sensitivity. This new MCV DNA detecting system is promising and we hope it can be coupled with histopathological and immunohistochemical studies to diagnose the viral status of MCC in clinics in the near future.

Gene Editing in Life Sciences

“…If the probe is in fact cut in response to Cas recognition of a COVID-specific sequence, biotin sticks to the negative band while FAM sticks to the positive band, showing a positive test result, displayed by the presence of a line at the FAM attachment location which is lacking in negative tests [7]. DETECTR can also be used to determine the presence of Merkel cell carcinoma, a form of skin cancer [8].…”