Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.
Keywords: CPER; SARS-CoV-2; infectious clone; mutagenesis; reverse genetics.
【저자키워드】 SARS-CoV-2, reverse genetics, Mutagenesis, CPER, infectious clone, 【초록키워드】 COVID-19, coronavirus disease, severe acute respiratory syndrome coronavirus 2, Coronavirus disease 2019, coronavirus, Mutation, Genome, severe acute respiratory syndrome Coronavirus, virus, genetics, PCR, cells, reporter viruses, reporter, assembly, Mutagenesis, Analysis, recombinant, mutant virus, causative agent, base pairs, acute respiratory syndrome, acute respiratory syndrome coronavirus, multiple mutations, construction, extension, transfection, high accuracy, mutant viruses, clone, bacterium, polymerase, SARS-CoV-2 cDNA, susceptible, SARS-CoV-2 infectious clones, DNA fragment, resulting, lack, generate, functional, rescued, multiple mutation, 【제목키워드】 genetics, extension, polymerase,