Adeno-Associated Virus-Mediated CRISPR-Cas13 Knockdown of Papain-like Protease from SARS-CoV-2 Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. SARS-CoV-2 Genome Bioinformatics Study
2.2. Cell Culture
2.3. Construction of PLpro Stable Cell Line
2.4. RNA Secondary Structures and Design of crRNA for CRISPR-Cas13 Systems
2.5. Compact CRISPR-Cas13 Plasmid Construction
2.6. Recombined Adeno-Associated Virus (rAAV) Production and Titration
2.7. rAAV Infection and Western Blotting
2.8. Total RNA Extraction and Reverse Transcriptase PCR (RT-PCR)
2.9. Real-Time Quantitative PCR (RT-qPCR)
2.10. Immunofluorescence (IF) Imaging
2.11. Statistical Analysis
3. Results
3.1. SARS-CoV-2 Antiviral Target Genome Mining
3.2. Structure-Based Design of crRNAs Simultaneously Targeting Three Distant Sites of PLpro mRNA
3.3. Compact CRISPR-Cas13 Knockdown PLpro with rAAV Vehicles
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Yang, Y.; Kessler, M.G.C.; Marchán-Rivadeneira, M.R.; Zhou, Y.; Han, Y. Adeno-Associated Virus-Mediated CRISPR-Cas13 Knockdown of Papain-like Protease from SARS-CoV-2 Virus. J 2024, 7, 393-405. https://doi.org/10.3390/j7030023
Yang Y, Kessler MGC, Marchán-Rivadeneira MR, Zhou Y, Han Y. Adeno-Associated Virus-Mediated CRISPR-Cas13 Knockdown of Papain-like Protease from SARS-CoV-2 Virus. J. 2024; 7(3):393-405. https://doi.org/10.3390/j7030023
Chicago/Turabian StyleYang, Yuehan, Mara Grace C. Kessler, M. Raquel Marchán-Rivadeneira, Yuxi Zhou, and Yong Han. 2024. "Adeno-Associated Virus-Mediated CRISPR-Cas13 Knockdown of Papain-like Protease from SARS-CoV-2 Virus" J 7, no. 3: 393-405. https://doi.org/10.3390/j7030023