A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning
Beom Kyu Kim, Won-Suk Choi, Ju Hwan Jeong, Sol Oh, Jihyun Park, Yu Soo Yun, Seong Cheol Min, Da Hyeon Kang, Eung‐Gook Kim, Hojin Ryu, Hye Kwon Kim, Yun Hee Baek, Young Ki Choi, Min‐Suk Song
Abstract
Since the first emergence of the SARS-CoV-2 variant in late 2020, novel variants have been continuously introduced to the human population, causing severe public health threats. In general, because these variants acquire new genetic mutation/s, it is critical to analyze the biological function of viruses that such mutations can confer. Therefore, we devised a method that can construct SARS-CoV-2 infectious clones and their variants rapidly and efficiently. The method was developed based on a PCR-based circular polymerase extension cloning (CPEC) combined with a specific primer design scheme. The efficiency of the newly designed method was evaluated by generating SARS-CoV-2 variants with single point mutations, multiple point mutations, and a large truncation and insertion. This method could be of value for the molecular characterization of emerging SARS-CoV-2 variants and the development and testing of vaccines and antiviral agents.