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      Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza

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          Summary

          The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.

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          Highlights

          • PAC-MAN is a CRISPR-based strategy for RNA-guided viral RNA inhibition and degradation

          • Cas13d PAC-MAC is effective at targeting and cleaving SARS-CoV-2 sequences

          • Cas13d PAC-MAC can reduce H1N1 IAV load in respiratory epithelial cells

          • A group of six crRNAs can target more than 90% of all coronaviruses

          Abstract

          A CRISPR-based strategy is developed to target conserved sequences across coronaviruses and other pathogenic viruses.

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          Most cited references32

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          A new coronavirus associated with human respiratory disease in China

          Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health 1–3 . Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing 4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China 5 . This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.
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            Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

            ABSTRACT A mysterious outbreak of atypical pneumonia in late 2019 was traced to a seafood wholesale market in Wuhan of China. Within a few weeks, a novel coronavirus tentatively named as 2019 novel coronavirus (2019-nCoV) was announced by the World Health Organization. We performed bioinformatics analysis on a virus genome from a patient with 2019-nCoV infection and compared it with other related coronavirus genomes. Overall, the genome of 2019-nCoV has 89% nucleotide identity with bat SARS-like-CoVZXC21 and 82% with that of human SARS-CoV. The phylogenetic trees of their orf1a/b, Spike, Envelope, Membrane and Nucleoprotein also clustered closely with those of the bat, civet and human SARS coronaviruses. However, the external subdomain of Spike’s receptor binding domain of 2019-nCoV shares only 40% amino acid identity with other SARS-related coronaviruses. Remarkably, its orf3b encodes a completely novel short protein. Furthermore, its new orf8 likely encodes a secreted protein with an alpha-helix, following with a beta-sheet(s) containing six strands. Learning from the roles of civet in SARS and camel in MERS, hunting for the animal source of 2019-nCoV and its more ancestral virus would be important for understanding the origin and evolution of this novel lineage B betacoronavirus. These findings provide the basis for starting further studies on the pathogenesis, and optimizing the design of diagnostic, antiviral and vaccination strategies for this emerging infection.
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              The spike protein of SARS-CoV — a target for vaccine and therapeutic development

              Key Points This Review provides an overview on the spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) as a target for the development of vaccines and therapeutics for the prevention and treatment of SARS. SARS is a newly emerging infectious disease, caused by SARS-CoV, a novel coronavirus that caused a global outbreak of SARS. SARS-CoV S protein mediates binding of the virus with its receptor angiotensin-converting enzyme 2 and promotes the fusion between the viral and host cell membranes and virus entry into the host cell. SARS-CoV S protein induces humoral and cellular immune responses against SARS-CoV. SARS S protein is the target of new SARS vaccines. These vaccines are based on SARS-CoV full-length S protein and its receptor-binding domain, including DNA-, viral vector- and subunit-based vaccines Peptides, antibodies, organic compounds and short interfering RNAs are additional anti-SARS-CoV therapeutics that target the S protein. The work on SARS-CoV S protein-based vaccines and drugs will be useful as a model for the development of prophylactic strategies and therapies against other viruses with class I fusion proteins that can cause emerging infectious diseases.
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                Author and article information

                Contributors
                Journal
                Cell
                Cell
                Cell
                Elsevier Inc.
                0092-8674
                1097-4172
                29 April 2020
                29 April 2020
                Affiliations
                [1 ]Department of Bioengineering, Stanford University, Stanford, CA 94305, USA
                [2 ]Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
                [3 ]Department of Management Science and Engineering, Stanford University, Stanford, CA 94305, USA
                [4 ]DNARx, San Francisco, CA 94107, USA
                [5 ]Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710, USA
                [6 ]Los Altos High School, Los Altos, CA 94022, USA
                [7 ]Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305, USA
                [8 ]ChEM-H, Stanford University, Stanford, CA 94305, USA
                Author notes
                []Corresponding author mlarussa@ 123456stanford.edu
                [∗∗ ]Corresponding author dblewis@ 123456stanford.edu
                [∗∗∗ ]Corresponding author stanley.qi@ 123456stanford.edu
                [9]

                These authors contributed equally

                [10]

                Lead Contact

                Article
                S0092-8674(20)30483-9
                10.1016/j.cell.2020.04.020
                7189862
                32353252
                07d007b4-cf1d-4cd8-9c99-9daec312e792
                © 2020 Elsevier Inc.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 11 March 2020
                : 24 March 2020
                : 13 April 2020
                Categories
                Article

                Cell biology
                crispr,cas13,covid-19,sars-cov-2,2019-ncov,rdrp,nucleocapsid,iav,influenza,antiviral
                Cell biology
                crispr, cas13, covid-19, sars-cov-2, 2019-ncov, rdrp, nucleocapsid, iav, influenza, antiviral

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