Rapid, field-deployable nucleobase detection and identification using FnCas9

Azhar, Mohd; Phutela, Rhythm; Ansari, Asgar Hussain; Sinha, Dipanjali; Sharma, Namrata; Kumar, Manoj; Aich, Meghali; Sharma, Saumya; Singhal, Khushboo; Lad, Harsha; Patra, Pradeep Kumar; Makharia, Govind; Chandak, Giriraj Ratan; Chakraborty, Debojyoti; Maiti, Souvik

doi:10.1101/2020.04.07.028167

Cited by 62 publications

(62 citation statements)

References 45 publications

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“…CRISPR-Cas nucleases can be easily programmed to target nucleic acids in a sequence-specific manner [19][20][21] , making them prime candidates for the detection and diagnosis of viral genetic material, and forming the CRISPR-based diagnostics (CRISPRDx) pipeline [22][23][24][25] . These systems rely on Type II Cas enzymes to physically bind target sequences 26 , or collateral cleavage by Type V or Type VI enzymes to detect DNA 24,25,27 or RNA species, respectively 22,23,28 . Since pandemic onset, an array of innovative diagnostics and prophylactics relying on these technologies have been adapted to detect or target SARS-CoV-2 with unprecedented speed 26,[29][30][31][32][33][34][35][36][37][38][39][40] , most notably represented by the DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) 24,25 and SHERLOCK (Specific High-Sensitivity Enzymatic Reporter unLOCKing) 22,23 systems (Summarized in Fig.…”

Section: Introductionmentioning

confidence: 99%

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

Brogan

Chaverra-Rodríguez

Lin

et al. 2020

Preprint

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Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

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Section: Introductionmentioning

confidence: 99%

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

Brogan

Chaverra-Rodríguez

Lin

et al. 2020

Preprint

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“…Next-generation point-of-care molecular diagnostic tools for COVID-19 can be developed using this method has the potential for the development of. Researchers in India have identified a Cas9 ortholog from Francisella novicida (FnCas9) that is very sensitive to nucleotide mismatches and developed the FnCas9 Editor Linked Uniform Detection Assay (FELUDA) for the diagnosis of SARS-CoV-2 infection in clinical settings as a point-of-care low-cost test (Azhar et al, 2020).…”

Section: Crispr-based Applications For Covid-19 Diagnosismentioning

confidence: 99%

CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics

Kumar

Malik

Ganesh

et al. 2020

Front. Cell. Infect. Microbiol.

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COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements.

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“…Utilizing the highly specific FnCas9 mediated DNA interrogation and subsequent cleavage, single nucleotide variant (SNV) detection system called FnCas9 Editor Linked Uniform Detection Assay (FELUDA) was developed by researchers in India. Recombinase Polymerase Amplification (RPA) was coupled with FELUDA to develop a field-deployable lateral flow assay targeting the NSP8 regions of SARS-CoV-2 for rapid diagnosis [35].…”

Section: Feludamentioning

confidence: 99%

Nucleic Acid and Immunological Diagnostics for SARS-CoV-2: Processes, Platforms and Pitfalls

Premraj

Aleyas

Nautiyal

et al. 2020

Preprint

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Accurate diagnosis at an early stage of infection is essential for the successful management of any contagious disease. The COVID-19, caused by the SARS-CoV-2 virus is a pandemic that has affected 214 countries affecting more than 30.8 million people causing 0.957 million deaths as of third week of September, 2020. The primary diagnosis of the infection is done either by the molecular technique of RT-qPCR by detecting portions of the RNA of the viral genome or through immunodiagnostic tests by detecting the viral proteins or the antibodies produced by the host. As the demand for the test increased rapidly many naive manufacturers entered the market with novel kits and more and more laboratories also entered the diagnostic arena making the test result more error-prone. There are serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on control strategies. The significance of the test is also complexed by the presence of asymptomatic carriers, re-occurrence of infection in cured patients as well as by the varied incubation periods of the infection and shifting of the viral location in the host tissues. In this paper, we review the techniques available for SARS-CoV-2 diagnosis and probable factors that can reduce the sensitivity and specificity of the different test methods currently in vogue. We also provide a check-list of factors to be taken care to avoid fallacious practices to reduce false positive and false negative results by the clinical laboratories

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Rapid, field-deployable nucleobase detection and identification using FnCas9

Cited by 62 publications

References 45 publications

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics

Nucleic Acid and Immunological Diagnostics for SARS-CoV-2: Processes, Platforms and Pitfalls

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