Design of CRISPR-Based Targets for the Development of a Diagnostic Method for SARS-CoV-2: An in Silico Approach
Objectives: To computationally design CRISPR target-based Single-guided RNA (sg RNA) for the development of a diagnostic method for SARS-CoV-2.
Methods: In the present study, Cas12 nuclease-specific protospacer adjacent motif (PAM) and downstream target sequences were identified from conserved regions of the SARS-CoV-2 spike glycoprotein S-gene receptor-binding domain (RBD) using CHOPCHOP and NEB computational tools to design sgRNA. Further, the in silico expression vector was constructed using SnapGene software.
Results: The 20-nucleotide sequence 5’-ACCATACAGAGTAGTAGTAC-3’ showed 100% similarity with SARS-CoV-2 was selected. Target-specific oligonucleotide sgRNA template was generated with the addition of an extra nucleotide ‘G’ to the 5’ end using NEBiocaluculator.
Conclusion: The findings of this study will help designing a CRISPR-based diagnostic kit for the detection of SARSCoV-2. However, computationally designed sgRNA requires to be tested in vitro to demonstrate its potential and efficiency in detecting this novel coronavirus.
1.Ishino Y, Krupovic M, Forterre P. History of CRISPR-Cas from Encounter with a Mysterious Repeated Sequence to Genome Editing Technology. J Bacteriol 2018;200:e00580–17.
2. Nasko DJ, Ferrell BD, Moore RM, Bhavsar JD, Polson SW, Wommack KE. CRISPR Spacers Indicate Preferential Matching of Specific Virioplankton Genes. mBio 2019;10:e02651–18.
3. Jiang F, Doudna JA. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys 2017;46:505–29.
4. Zhu Y, Klompe SE, Vlot M, van der Oost J, Staals RHJ. Shooting the messenger: RNA-targetting CRISPR-Cas systems. Biosci Rep 2018;38:BSR20170788.
5. Strich JR, Chertow DS. CRISPR-Cas Biology and Its Application to Infectious Diseases. J Clin Microbiol 2019;57:e01307–18.
6. Tang Y, Fu Y. Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing. Cell Biosci 2018;8:59.
7. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc 2019;14:2986–3012.
8. Yuen KS, Ye ZW, Fung SY, Chan CP, Jin DY. SARS-CoV-2 and COVID-19: The most important research questions. Cell Biosci 2020;10:40.
9. Udugama B, Kadhiresan P, Kozlowski HN, Malekjahani A, Osborne M, Li VYC, et al. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano 2020;14:3822–35.
10. Liu G, Zhang Y, Zhang T. Computational approaches for effective CRISPR guide RNA design and evaluation. Comput Struct Biotechnol J 2019;18:35–44.
11. Jaimes JA, André NM, Chappie JS, Millet JK, Whittaker GR. Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J Mol Biol 2020;432:3309–25.
12. Karlapudi AP, T C V, Tammineedi J, Srirama K, Kanumuri L, Prabhakar Kodali V. In silico sgRNA tool design for CRISPR control of quorum sensing in Acinetobacter species. Genes Dis 2018;5:123–9.
13. Abraham Peele K, Srihansa T, Krupanidhi S, Ayyagari VS, Venkateswarulu TC. Design of multi-epitope vaccine candidate against SARS-CoV-2: a in-silico study. J Biomol Struct Dyn. 2020 Jun 1:1-9. doi: 10.1080/07391102.2020.1770127. [Epub ahead of print].