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      Minimally instrumented SHERLOCK (miSHERLOCK) for CRISPR-based point-of-care diagnosis of SARS-CoV-2 and emerging variants

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          Abstract

          An integrated, low-cost, sample-to-answer, CRISPR-based diagnostic detects SARS-CoV-2 and variants from unprocessed saliva.

          Abstract

          The COVID-19 pandemic highlights the need for diagnostics that can be rapidly adapted and deployed in a variety of settings. Several SARS-CoV-2 variants have shown worrisome effects on vaccine and treatment efficacy, but no current point-of-care (POC) testing modality allows their specific identification. We have developed miSHERLOCK, a low-cost, CRISPR-based POC diagnostic platform that takes unprocessed patient saliva; extracts, purifies, and concentrates viral RNA; performs amplification and detection reactions; and provides fluorescent visual output with only three user actions and 1 hour from sample input to answer out. miSHERLOCK achieves highly sensitive multiplexed detection of SARS-CoV-2 and mutations associated with variants B.1.1.7, B.1.351, and P.1. Our modular system enables easy exchange of assays to address diverse user needs and can be rapidly reconfigured to detect different viruses and variants of concern. An adjunctive smartphone application enables output quantification, automated interpretation, and the possibility of remote, distributed result reporting.

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          Most cited references43

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          MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability

          We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
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            Jalview Version 2—a multiple sequence alignment editor and analysis workbench

            Summary: Jalview Version 2 is a system for interactive WYSIWYG editing, analysis and annotation of multiple sequence alignments. Core features include keyboard and mouse-based editing, multiple views and alignment overviews, and linked structure display with Jmol. Jalview 2 is available in two forms: a lightweight Java applet for use in web applications, and a powerful desktop application that employs web services for sequence alignment, secondary structure prediction and the retrieval of alignments, sequences, annotation and structures from public databases and any DAS 1.53 compliant sequence or annotation server. Availability: The Jalview 2 Desktop application and JalviewLite applet are made freely available under the GPL, and can be downloaded from www.jalview.org Contact: g.j.barton@dundee.ac.uk
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              Coronavirus biology and replication: implications for SARS-CoV-2

              The SARS-CoV-2 pandemic and its unprecedented global societal and economic disruptive impact has marked the third zoonotic introduction of a highly pathogenic coronavirus into the human population. Although the previous coronavirus SARS-CoV and MERS-CoV epidemics raised awareness of the need for clinically available therapeutic or preventive interventions, to date, no treatments with proven efficacy are available. The development of effective intervention strategies relies on the knowledge of molecular and cellular mechanisms of coronavirus infections, which highlights the significance of studying virus–host interactions at the molecular level to identify targets for antiviral intervention and to elucidate critical viral and host determinants that are decisive for the development of severe disease. In this Review, we summarize the first discoveries that shape our current understanding of SARS-CoV-2 infection throughout the intracellular viral life cycle and relate that to our knowledge of coronavirus biology. The elucidation of similarities and differences between SARS-CoV-2 and other coronaviruses will support future preparedness and strategies to combat coronavirus infections.
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                Author and article information

                Journal
                Sci Adv
                Sci Adv
                SciAdv
                advances
                Science Advances
                American Association for the Advancement of Science
                2375-2548
                August 2021
                06 August 2021
                : 7
                : 32
                : eabh2944
                Affiliations
                [1 ]Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA.
                [2 ]Institute for Medical Engineering and Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
                [3 ]Infectious Disease and Microbiome Program, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
                [4 ]Division of Infectious Diseases, Department of Pediatrics, Boston Children’s Hospital, Boston, MA 02115, USA.
                [5 ]Division of Infectious Diseases, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
                [6 ]Harvard Medical School, Boston, MA 02115, USA.
                [7 ]MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
                [8 ]Division of Gastroenterology, Massachusetts General Hospital, Boston, MA 02114, USA.
                [9 ]Abdul Latif Jameel Clinic for Machine Learning in Health, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
                [10 ]College of Arts and Sciences, Harvard University, Cambridge, MA 02138, USA.
                [11 ]Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
                [12 ]Harvard-MIT Program in Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
                Author notes
                [* ]Corresponding author. Email: jimjc@ 123456mit.edu
                [†]

                These authors contributed equally to this work and are listed in alphabetical order.

                Author information
                http://orcid.org/0000-0002-5368-6996
                http://orcid.org/0000-0001-9048-3592
                http://orcid.org/0000-0001-7890-7209
                http://orcid.org/0000-0001-5993-0091
                http://orcid.org/0000-0003-0826-904X
                http://orcid.org/0000-0002-0935-6404
                http://orcid.org/0000-0001-8782-3445
                http://orcid.org/0000-0002-7341-9860
                http://orcid.org/0000-0002-5560-8246
                Article
                abh2944
                10.1126/sciadv.abh2944
                8346217
                34362739
                014613ae-2d5c-4378-919f-6b2031170c22
                Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

                History
                : 01 March 2021
                : 17 June 2021
                Funding
                Funded by: doi http://dx.doi.org/10.13039/100016528, Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University;
                Funded by: MIT-Tata Center Fellowship;
                Funded by: Harvard University Center for AIDS Research (CFAR)/NIH;
                Award ID: P30 AI060354
                Funded by: Burroughs-Wellcome American Society of Tropical Medicine and Hygiene postdoctoral fellowship;
                Award ID: N/A
                Funded by: American Gastroenterological Association Takeda Pharmaceutical Research Scholar Award in Inflammatory Bowel Disease;
                Categories
                Research Article
                Research Articles
                SciAdv r-articles
                Health and Medicine
                Synthetic Biology
                Health and Medicine
                Custom metadata
                Nielsen Marquez

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