Team:LZU-CHINA/Description
At present, people all over the world is facing a pandemic of novel coronavirus pneumonia (COVID-19) caused by novel
coronavirus(SARS-CoV-2)[1]. The number of pademic cases and deaths worldwide is increasing, while traditional
vaccines and drug treatments are inevitably lagging behind. Therefore, in order to ensure human life safety,
this new threat makes people in urgen need of flexible and targeted protection measures. The potential of
CRISPR-Cas13d for novel coronavirus protection can provide us with new strategies to replace traditional
drugs or vaccines. According to recent research reports, several SARS-CoV-2 strains with different genome
sequences are spreading and evolving [2,3], which further highlights the need for targeted strategies of coronavirus.
Fig.1. Novel coronavirus (from National Institutes of Health)
SARS-CoV-2 is also known as a novel coronavirus because of its numerous granular processes at the edge of the virus particles. These granular processes are essentially a protein: Spike Protein (S protein). This protein is closely related to the mechanism of coronavirus infection in cells[2,3].
Fig.2. Novel coronavirus structure
SARS-CoV-2 relies on ACE2 on human cells in the process of entering cells [4,5].
Angiotensin-converting enzyme 2 (ACE2) is widely expressed in the renal, cardiovascular and gastrointestinal
systems in humans, and also expressed in alveolar epithelial cells. ACE2 is involved in biological processes
such as blood pressure lowering, coagulation system balance, nutrient absorption and intestinal inflammatory
reactions. Receptor-Binding Domain (RBD) of S protein binds to ACE2 on the surface of human cells through
conformational matching, and then enters cells.
Fig.3. S protein binding ACE2
In the process of virus infection, the spike protein binds to ACE2,
and the host cell secretes the corresponding protease to cut the
spike protein of the virus, releasing the fusion peptide and allowing
the virus to enter the host cells. The cell membrane then adsorbs and
internalizes the virus, and the virus enters the cytoplasm, where it
uncoats and releases its genome. After the genome is released into the
cytoplasm, the viral RNA binds to the ribosome of host cell and translates
into proteins. The proteins are then processed by protein hydrolase cleavage,
and then assembled into new viruses.
Fig.4. Hypothetical novel coronavirus life cycle
On the basis of the previous research on SARS-CoV-2, Prof.Dongqing Wei and Prof.Daixi Li of University of Shanghai for Science and Technology established an artificial intelligence drug screening platform, and screened out several potential novel coronavirus effective peptide inhibitors from a large number of candidate peptides. These peptide inhibitors are expected to inhibit the invasion of SARS-CoV-2 spike protein to human mucosal cells by binding to angiotensin converting enzyme 2 (ACE2) on human host cells, thereby blocking the viral transmission pathway. This also gives us new ideas for our experimental project: a way to identify and inhibit the expression levels of receptor proteins and related genes may be an effective treatment for COVID-19.
Does inhibition of ACE2 expression affect human health? We did a lot of research on that. Studies have found that the plateau becomes a protective factor against SARS-CoV-2, which may be due to the influence of the plateau environment on the cardiopulmonary system, thus inhibiting the ACE2 level on the cell surface[10,11]. However, the survey showed that low ACE2 level did not have a great impact on residents in plateau areas, indicating that the reduction of ACE2 expression would not cause significant structural abnormalities in the body[9].
Fig.5. Mortality rates of COVID-19 decreased with elevation[11]
How to knock down the expression level of ACE2? We chose CRISPR-Cas13d system. CRISPR-Cas13d is a RNA-guided CRISPR system targeting ssRNA. The system has been used to target viral sequences in human cells[15,16]. Cas13d uses CRISPR-related RNA (crRNA), which contains a customizable spacer sequence that can guide Cas13d protein to specific RNA molecules to target RNA degradation. Meanwhile, compared with other Cas13 proteins, Cas13d has the advantages of small volume (967 amino acids), high specificity and strong catalytic activity[18-20]. In addition, the RNA targeting cleavage activity of Cas13d was independent of the presence of specific adjacent sequences (PAM), which also prompted us to select Cas13d. The unique capabilities of the system meets the requirements of rapid development of gRNAs to target different viral variants that can evolve and may escape traditional drugs[21]. Therefore, the effect of CRISPR-Cas13d inhibiting the expression of receptor gene provides a potential mechanism for targeting SARS-CoV-2.
Fig.6. CRISPR-Cas13 system
Fig.7. Protection of Pancoronavirus by CRISPR[13]
References
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[2]X. Tang et al., On the origin and continuing evolution of SARS-CoV-2. National Science Review, (2020).
[3]L. Du et al., The spike protein of SARS-CoV — a target for vaccine and therapeutic development. Nature Reviews Microbiology 7, 226-236 (2009).
[4]H. Zhang et al., Digestive system is a potential route of COVID-19: an analysis of single-cell coexpression pattern of key proteins in viral entry process. Gut 69, 1010 (2020).
[5]M. Hoffmann et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, (2020).
[6]V. Shenoy et al., The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension. Am J Respir Crit Care Med 182, 1065-1072 (2010).
[7]D. Singer et al., Defective intestinal amino acid absorption in Ace2 null mice. American Journal of Physiology-gastrointestinal and Liver Physiology 303, (2012).
[8]T. Yang et al., Gnotobiotic Rats Reveal That Gut Microbiota Regulates Colonic mRNA of Ace2, the Receptor for SARS-CoV-2 Infectivity. Hypertension (Dallas, Tex. : 1979) 76, e1-e3 (2020).
[9]S. B. Gurley et al., Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. The Journal of Clinical Investigation 116, 2218-2225 (2006).
[10]V. Shenoy, Y. Qi, M. J. Katovich, M. K. Raizada, ACE2, a promising therapeutic target for pulmonary hypertension. Current Opinion in Pharmacology 11, 150-155 (2011).
[11]T. M. Thomson et al., Altitude as a protective factor from COVID-19. medRxiv, 2020.2008.2003.20167262 (2020).
[12]X. Ou et al., Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nature communications 11, 1620 (2020).
[13]T. R. Abbott et al., Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza. Cell 181, 865-876.e812 (2020).
[14]T. M. Nguyen, Y. Zhang, P. P. Pandolfi, Virus against virus: a potential treatment for 2019-nCov (SARS-CoV-2) and other RNA viruses. Cell Research 30, 189-190 (2020).
[15]S. Bawage, P. M. Tiwari, P. J. Santangelo, Synthetic mRNA expressed Cas13a mitigates RNA virus infections. bioRxiv, 370460 (2018).
[16]C. A. Freije et al., Programmable Inhibition and Detection of RNA Viruses Using Cas13. Molecular Cell 76, 826 (2019).
[17]S. Konermann et al., Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell 173, 665-676 (2018).
[18]W. X. Yan et al., Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein. Molecular Cell 70, (2018).
[19]J. S. Gootenberg et al., Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360, 439-444 (2018).
[20]A. Smargon et al., Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28. Molecular Cell 65, 618-630 (2017).
[21]S. Konermann et al., Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell 173, 665-676.e614 (2018).
