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; Ji Hyun 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

doi:10.1128/spectrum.03385-22

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, Ji Hyun 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

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

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, Δ69-70, Δ157-158, E484K1N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) 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.

Original language	English
Journal	Microbiology Spectrum
Volume	11
Issue number	2
DOIs	https://doi.org/10.1128/spectrum.03385-22
State	Published - Mar 2023
Externally published	Yes

Keywords

CPEC
infectious clone
mutagenesis
reverse genetics
SARS-CoV-2

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1128/spectrum.03385-22

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Kim, B. K., Choi, W. S., Jeong, J. H., Oh, S., Park, J. H., Yun, Y. S., Min, S. C., Kang, D. H., Kim, E. G., Ryu, H., Kim, H. K., Baek, Y. H., Choi, Y. K., & Song, M. S. (2023). A Rapid Method for Generating Infectious SARS-CoV-2 and Variants Using Mutagenesis and Circular Polymerase Extension Cloning. Microbiology Spectrum, 11(2). https://doi.org/10.1128/spectrum.03385-22

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

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, Δ69-70, Δ157-158, E484K1N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) 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.",

keywords = "CPEC, infectious clone, mutagenesis, reverse genetics, SARS-CoV-2",

author = "Kim, \{Beom Kyu\} and Choi, \{Won Suk\} and Jeong, \{Ju Hwan\} and Sol Oh and Park, \{Ji Hyun\} and Yun, \{Yu Soo\} and Min, \{Seong Cheol\} and Kang, \{Da Hyeon\} and Kim, \{Eung Gook\} and Hojin Ryu and Kim, \{Hye Kwon\} and Baek, \{Yun Hee\} and Choi, \{Young Ki\} and Song, \{Min Suk\}",

note = "Publisher Copyright: Copyright {\textcopyright} 2023 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.",

year = "2023",

month = mar,

doi = "10.1128/spectrum.03385-22",

language = "English",

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AU - Choi, Won Suk

AU - Jeong, Ju Hwan

AU - Oh, Sol

AU - Park, Ji Hyun

AU - Yun, Yu Soo

AU - Min, Seong Cheol

AU - Kang, Da Hyeon

AU - Kim, Eung Gook

AU - Ryu, Hojin

AU - Kim, Hye Kwon

AU - Baek, Yun Hee

AU - Choi, Young Ki

AU - Song, Min Suk

PY - 2023/3

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N2 - 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, Δ69-70, Δ157-158, E484K1N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) 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.

AB - 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, Δ69-70, Δ157-158, E484K1N501Y, and Ins-38F) and multiple mutations (N501Y/D614G and E484K/N501Y/D614G), as well as a large truncation (ΔORF7A) 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.

KW - CPEC

KW - infectious clone

KW - mutagenesis

KW - reverse genetics

KW - SARS-CoV-2

UR - https://www.scopus.com/pages/publications/85154068710

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DO - 10.1128/spectrum.03385-22

M3 - Article

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AN - SCOPUS:85154068710

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JO - Microbiology Spectrum

JF - Microbiology Spectrum

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ER -

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

Abstract

Keywords

UN SDGs

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