Cloning, assembly, and modification of the primary human cytomegalovirus isolate Toledo by yeast-based transformation-associated recombination

Sanjay Vashee, Timothy B. Stockwell, Nina Alperovich, Evgeniya A. Denisova, Daniel G. Gibson, Kyle C. Cady, Kristofer Miller, Krishna Kannan, Daniel Malouli, Lindsey B. Crawford, Alexander A. Voorhies, Eric Bruening, Patrizia Caposio, Klaus Frueh

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA.

Original languageEnglish (US)
Article numbere00331-17
JournalmSphere
Volume2
Issue number5
DOIs
StatePublished - Sep 1 2017

Fingerprint

Cytomegalovirus
Genetic Recombination
Organism Cloning
Yeasts
Genome
Bacterial Artificial Chromosomes
Fibroblasts
Viral Genome
Saccharomyces cerevisiae
Clone Cells
Synthetic Biology
Replication Origin
Tropism
Genetic Engineering
Viral Genes
Sequence Deletion
DNA
Sequence Analysis
Cell Culture Techniques
Viruses

Keywords

  • Cloning
  • Cytomegalovirus
  • Genetic recombination
  • Saccharomyces cerevisiae

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Cloning, assembly, and modification of the primary human cytomegalovirus isolate Toledo by yeast-based transformation-associated recombination. / Vashee, Sanjay; Stockwell, Timothy B.; Alperovich, Nina; Denisova, Evgeniya A.; Gibson, Daniel G.; Cady, Kyle C.; Miller, Kristofer; Kannan, Krishna; Malouli, Daniel; Crawford, Lindsey B.; Voorhies, Alexander A.; Bruening, Eric; Caposio, Patrizia; Frueh, Klaus.

In: mSphere, Vol. 2, No. 5, e00331-17, 01.09.2017.

Research output: Contribution to journalArticle

Vashee, S, Stockwell, TB, Alperovich, N, Denisova, EA, Gibson, DG, Cady, KC, Miller, K, Kannan, K, Malouli, D, Crawford, LB, Voorhies, AA, Bruening, E, Caposio, P & Frueh, K 2017, 'Cloning, assembly, and modification of the primary human cytomegalovirus isolate Toledo by yeast-based transformation-associated recombination', mSphere, vol. 2, no. 5, e00331-17. https://doi.org/10.1128/mSphere.00331-17
Vashee, Sanjay ; Stockwell, Timothy B. ; Alperovich, Nina ; Denisova, Evgeniya A. ; Gibson, Daniel G. ; Cady, Kyle C. ; Miller, Kristofer ; Kannan, Krishna ; Malouli, Daniel ; Crawford, Lindsey B. ; Voorhies, Alexander A. ; Bruening, Eric ; Caposio, Patrizia ; Frueh, Klaus. / Cloning, assembly, and modification of the primary human cytomegalovirus isolate Toledo by yeast-based transformation-associated recombination. In: mSphere. 2017 ; Vol. 2, No. 5.
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abstract = "Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA.",
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AU - Vashee, Sanjay

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AU - Denisova, Evgeniya A.

AU - Gibson, Daniel G.

AU - Cady, Kyle C.

AU - Miller, Kristofer

AU - Kannan, Krishna

AU - Malouli, Daniel

AU - Crawford, Lindsey B.

AU - Voorhies, Alexander A.

AU - Bruening, Eric

AU - Caposio, Patrizia

AU - Frueh, Klaus

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N2 - Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA.

AB - Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA.

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