![]() ![]() ![]() In addition, our approach differs from previous studies in that we aim to develop a strategy for simultaneously targeting multiple species of viruses within the same family to provide broad, pan-coronavirus protection. While this is not the first study to use a Cas13 variant to target viral sequences in human cells ( Bawage et al., 2018, Freije et al., 2019), it remains unknown whether CRISPR tools can be designed to effectively target and cleave SARS-CoV-2 sequences. Because of its small size (967 amino acids), high specificity, and strong catalytic activity, we chose Cas13d rather than other Cas13 proteins to target and destroy RNA viruses, including SARS-CoV-2 and influenza A virus (IAV) ( Gootenberg et al., 2018, Smargon et al., 2017, Yan et al., 2018). The high catalytic activity of Cas13d in human cells provides a potential mechanism for targeting SARS-CoV-2 for specific viral RNA genome degradation and viral gene expression inhibition. Cas13d employs CRISPR-associated RNAs (crRNAs) that contain a customizable 22-nt spacer sequence that can direct the Cas13d protein to specific RNA molecules for targeted RNA degradation. To inhibit RNA viruses in human cells, we used the class 2 type VI-D CRISPR-Cas13d system derived from Ruminococcus flavefaciens XPD3002, a recently discovered RNA-guided RNA endonuclease ( Konermann et al., 2018, Yan et al., 2018). (B) Cas13d can inhibit viral function and replication by directly targeting and cleaving all viral positive-sense RNA. Upon SARS-CoV-2 entry and genome RNA release, the positive strand RNA genome serves as a template to make negative strand genomic and subgenomic templates, which are used to produce more copies of the positive strand viral genome and viral mRNAs. (A) A hypothetical life cycle of SARS-CoV-2. The Hypothetical Life Cycle of SARS-CoV-2 and the PAC-MAN Approach for Inhibiting Coronavirus Using CRISPR-Cas13 Targeting the positive-sense genome and viral mRNAs to simultaneously degrade viral genome templates for replication and viral gene expression would be expected to robustly limit viral replication. ![]() Whereas most ongoing vaccine trials work by priming the human immune system to recognize coronavirus proteins or attenuated viruses and decrease viral entry into cells ( Rappuoli, 2018), the alternative antiviral approach we propose here relies on a CRISPR-based system for recognizing and degrading the intracellular viral genome and its resulting viral mRNAs ( Figure 1B). The SARS-CoV-2 life cycle is likely similar to the closely related coronavirus that causes SARS, in which the virus enters the cell, releases its RNA genome into the cytoplasm, and synthesizes negative-sense genomic and subgenomic RNAs from which viral mRNAs and new copies of the positive sense viral genome are synthesized ( Figure 1Ī) ( Du et al., 2009, Liu, 2014). The novel coronavirus causing COVID-19 belongs to a family of positive-sense RNA viruses, which typically infect the upper and lower respiratory tracts and cause disease by direct cytotoxic effects and the induction of host cytokine-mediated inflammation ( Liu et al., 2020). A recent report showed two strains (L and S) of SARS-CoV-2 with different genomic sequences are circulating and likely evolving, further highlighting the need for a pan-coronavirus targeting strategy ( Tang et al., 2020). Because coronaviruses causing COVID-19, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS) are able to spontaneously transfer to humans from diverse animal hosts that act as viral reservoirs, there is a pressing need to develop methods to broadly combat other coronaviruses that may emerge in the future ( Li et al., 2005, Mohd et al., 2016, Shi and Hu, 2008). With a rapidly growing number of cases and deaths around the world, such emerging threats require a nimble and targeted means of protection. It is predicted that the development of a safe and effective vaccine to prevent COVID-19 will take 12 to 18 months, by which time many millions of people may have been infected. The world is currently faced with a pandemic of novel coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has no preventative vaccine or proven pharmacologic treatment.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |