Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

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 inefficie...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	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 Früh
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Saccharomyces cerevisiae cloning cytomegalovirus genetic recombination Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a21d363ce75f422ea5457758aa820bd9
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:a21d363ce75f422ea5457758aa820bd9
record_format	dspace
spelling	oai:doaj.org-article:a21d363ce75f422ea5457758aa820bd92021-11-15T15:22:05ZCloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination10.1128/mSphereDirect.00331-172379-5042https://doaj.org/article/a21d363ce75f422ea5457758aa820bd92017-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphereDirect.00331-17https://doaj.org/toc/2379-5042ABSTRACT 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. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.Sanjay VasheeTimothy B. StockwellNina AlperovichEvgeniya A. DenisovaDaniel G. GibsonKyle C. CadyKristofer MillerKrishna KannanDaniel MalouliLindsey B. CrawfordAlexander A. VoorhiesEric BrueningPatrizia CaposioKlaus FrühAmerican Society for MicrobiologyarticleSaccharomyces cerevisiaecloningcytomegalovirusgenetic recombinationMicrobiologyQR1-502ENmSphere, Vol 2, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Saccharomyces cerevisiae cloning cytomegalovirus genetic recombination Microbiology QR1-502
spellingShingle	Saccharomyces cerevisiae cloning cytomegalovirus genetic recombination Microbiology QR1-502 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 Früh Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
description	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. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.
format	article
author	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 Früh
author_facet	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 Früh
author_sort	Sanjay Vashee
title	Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
title_short	Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
title_full	Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
title_fullStr	Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
title_full_unstemmed	Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination
title_sort	cloning, assembly, and modification of the primary human cytomegalovirus isolate toledo by yeast-based transformation-associated recombination
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/a21d363ce75f422ea5457758aa820bd9
work_keys_str_mv	AT sanjayvashee cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT timothybstockwell cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT ninaalperovich cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT evgeniyaadenisova cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT danielggibson cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT kyleccady cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT kristofermiller cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT krishnakannan cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT danielmalouli cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT lindseybcrawford cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT alexanderavoorhies cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT ericbruening cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT patriziacaposio cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination AT klausfruh cloningassemblyandmodificationoftheprimaryhumancytomegalovirusisolatetoledobyyeastbasedtransformationassociatedrecombination
_version_	1718428088288673792

Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination

Ejemplares similares