Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform

被引：331

作者：

Tran Thi Nhu Thao ^{[1
,2
,3
]}

Labroussaa, Fabien ^{[2
,4
]}

Ebert, Nadine ^{[1
,2
]}

V'kovski, Philip ^{[1
,2
]}

Stalder, Hanspeter ^{[1
,2
]}

Portmann, Jasmine ^{[1
,2
]}

Kelly, Jenna ^{[1
,2
]}

Steiner, Silvio ^{[1
,2
,3
]}

Holwerda, Melle ^{[1
,2
,3
,5
]}

Kratzel, Annika ^{[1
,2
,3
]}

Gultom, Mitra ^{[1
,2
,3
,5
]}

Schmied, Kimberly ^{[1
,2
]}

Laloli, Laura ^{[1
,2
,3
,5
]}

Huesser, Linda ^{[1
,2
]}

Wider, Manon ^{[5
]}

Pfaender, Stephanie ^{[1
,2
,6
]}

Hirt, Dagny ^{[1
,2
]}

Cippa, Valentina ^{[2
,4
]}

Crespo-Pomar, Silvia ^{[2
,4
]}

Schroeder, Simon ^{[7
,8
,9
,10
]}

Muth, Doreen ^{[7
,8
,9
,10
,11
]}

Niemeyer, Daniela ^{[7
,8
,9
,10
,11
]}

Corman, Victor M. ^{[7
,8
,9
,10
,11
]}

Mueller, Marcel A. ^{[7
,8
,9
,10
,11
,12
]}

Drosten, Christian ^{[7
,8
,9
,10
,11
]}

Dijkman, Ronald ^{[1
,2
,5
]}

Jores, Joerg ^{[2
,4
]}

Thiel, Volker ^{[1
,2
]}

机构：

[1] IVI, Bern, Switzerland

[2] Univ Bern, Vetsuisse Fac, Dept Infect Dis & Pathobiol, Bern, Switzerland

[3] Univ Bern, Grad Sch Biomed Sci, Bern, Switzerland

[4] Univ Bern, Vetsuisse Fac, Inst Vet Bacteriol, Bern, Switzerland

[5] Univ Bern, Inst Infect Dis, Bern, Switzerland

[6] Ruhr Univ Bochum, Dept Mol & Med Virol, Bochum, Germany

[7] Charite Univ Med Berlin, Inst Virol, Berlin, Germany

[8] Free Univ Berlin, Berlin, Germany

[9] Humboldt Univ, Berlin, Germany

[10] Berlin Inst Hearth, Berlin, Germany

[11] Associated Partner Charite, German Ctr Infect Res, Berlin, Germany

[12] Sechenov Univ, Martsinovsky Inst Med Parasitol Trop & Vector Bor, Moscow, Russia

来源：

NATURE | 2020年 / 582卷 / 7813期

基金：

瑞士国家科学基金会;

关键词：

INFECTIOUS CLONE; IN-VITRO; CORONAVIRUS; RNA; SYSTEM;

D O I：

10.1038/s41586-020-2294-9

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate inEscherichia coliowing to the size and occasional instability of the genome(1-3). Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of theCoronaviridae,FlaviviridaeandPneumoviridaefamilies. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step inSaccharomyces cerevisiaeusing transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(4), which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak. A yeast-based synthetic genomics platform is used to reconstruct and characterize large RNA viruses from synthetic DNA fragments; this technique will facilitate the rapid analysis of RNA viruses, such as SARS-CoV-2, during an outbreak.

引用

页码：561 / +

页数：21

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