Life science
Infectious diseases

A novel, quick, and easy system for genetic analysis ofSARS-CoV-2
Center for Infectious Disease Education and Research, Research Institute for Microbial Diseases
SA Professor Yoshiharu Matsuura

Researchmap https://researchmap.jp/matsuura_123

SA Associate Professor Chikako Ono

Researchmap https://researchmap.jp/ChikakoOno

Life science

Abstract
Our group has established a novel PCR-based, bacterium-freereverse genetics system for SARS-CoV-2 using the CPER method.
This method is simpler and faster than previous methods. Large
numbers of recombinant SARS-CoV-2 can be produced in about
two weeks. This method can be used to modify the viral genome,
allowing detailed study of the functions of mutations and the pathogenesis of COVID-19, and could speed the development of preventative measures and therapeutic strategies for the disease.

▍ Background & Results
SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. We know that mutations in the genome of SARS-CoV-2 have occurred and spread, but what effect do those mutations have? Current methods for studying mutations in the SARS-CoV-2 genome
are very complicated and time-consuming because coronaviruseshave large genomes, but now a team from Osaka University andHokkaido University have developed a quick, PCR-based reverse
genetics system for analyzing SARS-CoV-2 mutations.
This system uses the polymerase chain reaction (PCR) and acircular polymerase extension reaction (CPER) to reconstruct thefull-length cDNA of viral genome. This process does not involve theuse of bacteria, which can introduce further unwanted mutations,and takes only two weeks using simple steps to generate infectious
virus particles. Previous methods took a couple of months andwere very complicated procedures.

▍ Significance of the research and Future perspective
This method allows us to quickly examine the biological featuresof mutations in the SARS-CoV-2. The CPER technique can be used
to create recombinant viruses with each mutation and examine
their biological features in comparison with the parental virus andcontribute to understand the mechanisms underlying propagationand pathogenesis of SARS-CoV-2, as well as help determine thebiological significance of emerging mutations. This method could
could even allow a recombinant virus that is unable to cause disease to be generated, which could be used as a safe and effective
vaccine for SARS-CoV-2.
Mutations are arising in the SARS-CoV-2 population all the time,
as well as questions as to what those mutations do and whether
they could affect the efficacy of vaccines. This simple and rapid
method allows scientists around the globe to characterize the mutants, which is a vital step forward in our fight against the SARSCoV-2.

Figure. Reverse genetics for SARS-CoV-2 by CPER method In total of 9 genefragments covering the full-length SARS-Cov-2 genome and a linker fragment
were amplified. Since all fragments were designed to include overlapping ends
with adjacent fragments, they can be assembled as a circular viral genome byadditional PCR. By transfection of the circular viral genome into the susceptible
cells, recombinant SARS-CoV-2 were rescued. Cytopathic effects were observed
only in the cells transfected with CPER products.

P a t e n t
Torii, Shiho; Ono, Chikako; Suzuki, Rigel et al. Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction.
Cell Rep. 2021 Apr; 35(3): 109014. doi: 10.1016/j.celrep.2021.109014. Epub 2021 Apr 1.
UR
L http://www.biken.osaka-u.ac.jp/achievement/research/2021/152
K e y w o r d SARS-CoV-2, CPER, PCR
Tr e a t i s e