Next-Generation Sequencing and CRISPR/Cas13 Editing in Viroid Research and Molecular Diagnostics

Hadidi, A.

doi:10.3390/v11020120

Cited by 48 publications

(29 citation statements)

References 101 publications

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“…Platforms such as DETECTR by Jennifer Doudna's group (Chen et al, 2018) and SHERLOCK (Gootenberg et al, 2017(Gootenberg et al, , 2018 by Feng Zhang's group have leveraged this search functionality to get sensitive and specific diagnostic results for HPV and Dengue virus, respectively. Further studies to use CRISPR for Zika virus, Lassa fever, and even Ebola are underway (Chertow, 2018;Pan and Kraschel, 2018;Hadidi, 2019;Pickar-Oliver and Gersbach, 2019). The startups called Mammoth Biosciences and SHERLOCK Biosciences are such endeavors for providing better diagnostics for disease.…”

Section: Discussionmentioning

confidence: 99%

DETECTing Merkel Cell Polyomavirus in Merkel Tumors

Arora

Gupta

Vijaykumar

et al. 2020

Front. Mol. Biosci.

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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 MCV-positive Merkel cell carcinoma patients is better, making this differential information of significant diagnostic and prognostic value. Also, MCV provides a direct target for therapy in MCC patients. Currently, the methods used for diagnosis of MCV in tumors are often discordant and unreliable. Here 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, 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 MCV DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and with femto-molar sensitivity. Our study is a preliminary, proof-of-principle analysis showing the use of CRISPR for MCV diagnosis. Further validation in human tumor samples is needed for its clinical use in the near future. This new system is promising and we hope it can be coupled with immunohistochemical studies to diagnose the viral status of MCC in clinics soon.

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Section: Discussionmentioning

confidence: 99%

DETECTing Merkel Cell Polyomavirus in Merkel Tumors

Arora

Gupta

Vijaykumar

et al. 2020

Front. Mol. Biosci.

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“…Platforms such as DETECTR by Jennifer Doudna's group 29 and SHERLOCK 42,43 by Feng Zhang's group have leveraged this search functionality to get sensitive and specific diagnostic results for HPV and Dengue virus respectively. Further studies to use CRISPR for Zika virus, Lassa fever and even Ebola are underway [44][45][46][47] . The startups called Mammoth Biosciences and SHERLOCK Biosciences are such endeavors for providing better diagnostics for disease.…”

Section: Discussionmentioning

confidence: 99%

DETECTing Merkel cell Polyomavirus in Merkel Tumours

Arora

Gupta

Vijaykumar

et al. 2019

Preprint

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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.

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“…NGS has been widely used in viroid research for the discovery of new viroids and diagnostic purposes. The technology has been employed to analyze viroid sequences and host gene expression (transcriptomic) in response to viroid infection [ 47 ]. Progressive filtering of overlapping small RNAs-1/-2 (PROF1/PROF2) uses deeply sequenced small RNAs and assembles them into circular RNAs representing possible viroids and satellite RNAs.…”

Section: Viroidsmentioning

confidence: 99%

Long Noncoding RNAs in Plant Viroids and Viruses: A Review

Shrestha

Bujarski

2020

Pathogens

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Infectious long-noncoding (lnc) RNAs related to plants can be of both viral and non-viral origin. Viroids are infectious plant lncRNAs that are not related to viruses and carry the circular, single-stranded, non-coding RNAs that replicate with host enzymatic activities via a rolling circle mechanism. Viroids interact with host processes in complex ways, emerging as one of the most productive tools for studying the functions of lncRNAs. Defective (D) RNAs, another category of lnc RNAs, are found in a variety of plant RNA viruses, most of which are noncoding. These are derived from and are replicated by the helper virus. D RNA-virus interactions evolve into mutually beneficial combinations, enhancing virus fitness via competitive advantages of moderated symptoms. Yet the satellite RNAs are single-stranded and include either large linear protein-coding ss RNAs, small linear ss RNAs, or small circular ss RNAs (virusoids). The satellite RNAs lack sequence homology to the helper virus, but unlike viroids need a helper virus to replicate and encapsidate. They can attenuate symptoms via RNA silencing and enhancement of host defense, but some can be lethal as RNA silencing suppressor antagonists. Moreover, selected viruses produce lncRNAs by incomplete degradation of genomic RNAs. They do not replicate but may impact viral infection, gene regulation, and cellular functions. Finally, the host plant lncRNAs can also contribute during plant-virus interactions, inducing plant defense and the regulation of gene expression, often in conjunction with micro and/or circRNAs.

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Next-Generation Sequencing and CRISPR/Cas13 Editing in Viroid Research and Molecular Diagnostics

Cited by 48 publications

References 101 publications

DETECTing Merkel Cell Polyomavirus in Merkel Tumors

DETECTing Merkel Cell Polyomavirus in Merkel Tumors

DETECTing Merkel cell Polyomavirus in Merkel Tumours

Long Noncoding RNAs in Plant Viroids and Viruses: A Review

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