BROWSE

ITEM VIEW & DOWNLOAD

A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning

Cited 0 time in webofscience Cited 0 time in scopus
64 Viewed 0 Downloaded
Title
A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning
Author(s)
Kim, Beom Kyu; Choi, Won-Suk; Jeong, Ju Hwan; Oh, Sol; Park, Ji-Hyun; Yun, Yu Soo; Min, Seong Cheol; Kang, Da Hyeon; Kim, Eung-Gook; Ryu, Hojin; Kim, Hye Kwon; Baek, Yun Hee; Choi, Young Ki; Song, Min-Suk
Publication Date
2023-04
Journal
Microbiology spectrum, v.11, no.2
Publisher
American Society for Microbiology
Abstract
The appearance of SARS-CoV-2 variants in late 2020 raised alarming global public health concerns. Despite continued scientific progress, the genetic profiles of these variants bring changes in viral properties that threaten vaccine efficacy. Thus, it is critically important to investigate the biologic profiles and significance of these evolving variants. In this study, we demonstrate the application of circular polymerase extension cloning (CPEC) to the generation of full-length clones of SARS-CoV-2. We report that, combined with a specific primer design scheme, this yields a simpler, uncomplicated, and versatile approach for engineering SARS-CoV-2 variants with high viral recovery efficiency. This new strategy for genomic engineering of SARS-CoV-2 variants was implemented and evaluated for its efficiency in generating point mutations (K417N, L452R, E484K, N501Y, D614G, P681H, P681R, Delta 69-70, Delta 157-158, E484K+N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (Delta ORF7A) and insertion (GFP). The application of CPEC to mutagenesis also allows the inclusion of a confirmatory step prior to assembly and transfection. This method could be of value in the molecular characterization of emerging SARS-CoV-2 variants as well as the development and testing of vaccines, therapeutic antibodies, and antivirals.IMPORTANCE 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. 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.
URI
https://pr.ibs.re.kr/handle/8788114/14007
DOI
10.1128/spectrum.03385-22
ISSN
2165-0497
Appears in Collections:
Korea Virus Research Institute(한국바이러스기초연구소) > Center for Study of Emerging and Re-emerging Viruses(신변종 바이러스 연구센터) > 1. Journal Papers (저널논문)
Files in This Item:
There are no files associated with this item.

qrcode

  • facebook

    twitter

  • Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.
해당 아이템을 이메일로 공유하기 원하시면 인증을 거치시기 바랍니다.

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse