ABSTRACT
Because of the essential roles of SARS-CoV-2 papain-like protease (PLpro) in the viral polyprotein processing and suppression of host immune responses, it is a crucial target for drug discovery against COVID-19. To develop robust biochemical methodologies for inhibitor screening against PLpro, extensive characterization of recombinant protein is important. Here we report cloning, expression, and purification of the recombinant SARS-CoV-2 PLpro, and explore various parameters affecting its stability and the catalytic activity. We also report the optimum conditions which should be used for high-throughput inhibitor screening using a fluorogenic tetrapeptide substrate.
Subject(s)
Coronavirus Papain-Like Proteases/chemistry , Coronavirus Papain-Like Proteases/metabolism , High-Throughput Screening Assays/methods , Antiviral Agents/pharmacology , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/isolation & purification , Coumarins/chemistry , Coumarins/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Dimethyl Sulfoxide/chemistry , Dynamic Light Scattering , Edetic Acid/chemistry , Enzyme Stability , Fluorometry/methods , Hydrogen-Ion Concentration , Osmolar Concentration , Peptides/chemistry , Peptides/metabolism , TemperatureABSTRACT
Coronavirus disease 2019 (COVID-19), an infectious disease caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a persistent global threat. The transmission of SARS-CoV-2 is wide and swift. Rapid detection of the viral RNA and effective therapy are imperative to prevent the worldwide spread of the new infectious disease. Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)- CRISPR-associated protein (Cas) system is an RNA-directed adaptive immune system, and it has been transformed into a gene editing tool. Applications of CRISPR-Cas system involves in many fields, such as human gene therapy, drug discovery and disease diagnosis. Under the background of COVID-19 pandemic, CRISPR-Cas system shows hidden capacity to fight the emergency in many aspects. This review will focus on the role of gene editing in COVID-19 diagnosis and treatment. We will describe the potential use of CRISPR-Cas-based system in combating COVID-19, from diagnosis to treatment. Furthermore, the limitation and perspectives of this novel technology are also evaluated.
Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , COVID-19/therapy , CRISPR-Cas Systems , Gene Editing/methods , Gene Expression Regulation, Viral/genetics , RNA, Viral/analysis , SARS-CoV-2/genetics , Animals , Fluorometry/methods , Forecasting , Gene Knockout Techniques , High-Throughput Nucleotide Sequencing , Host-Pathogen Interactions , Humans , Mice , Mice, Transgenic , Models, Animal , Molecular Targeted Therapy , Nasopharynx/virology , Oropharynx/virology , RNA, Viral/genetics , RNA, Viral/metabolism , Real-Time Polymerase Chain Reaction , SARS-CoV-2/isolation & purification , Sensitivity and SpecificityABSTRACT
The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.