Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.
Cloning, Assembly, and Modification of the Primary Human Cytomegalovirus Isolate Toledo by Yeast-Based Transformation-Associated Recombination.
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DOI：
10.1128/mspheredirect.00331-17
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发表时间：
2017-09
期刊：
影响因子：
4.8
通讯作者：
Früh K
中科院分区：
文献类型：
--
作者：
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;Früh K
大DNA病毒如人巨细胞病毒（HCMV）的基因组难以使用当前的遗传工具进行操作，并且此时，不进行广泛的组织培养不可能获得CMV的分子克隆。为了克服这些限制，我们使用合成生物学工具从病毒DNA中捕获基因组片段，并在酵母中组装全长基因组。使用含有野生型和组织培养适应病毒的混合物的HCMV分离株Toledo的早期传代。我们直接克隆了多数序列，并通过同时修饰多个基因组区域来重建少数序列。因此，我们的新方法提供了一种范例，不仅可以在全基因组范围内有效地工程化HCMV和其他大型DNA病毒，还可以促进原代分离株的克隆和遗传操作，并提供了一种生成完全合成基因组的途径。巨细胞病毒（CMV）的基因工程目前依赖于通过在病毒感染的组织培养细胞中使用体内重组将细菌复制起点引入病毒基因组来产生细菌人工染色体（BAC）。然而，该过程是低效的，导致适应性突变，并且涉及病毒基因的缺失以避免在插入BAC盒时过大的基因组。此外，BAC技术不允许同时操作多个基因组基因座，并且不能用于构建合成基因组。为了克服这些限制，我们采用合成生物学工具在酿酒酵母中克隆CMV基因组。使用人CMV分离株Toledo的早期传代，我们首先应用转化相关重组（TAR）在酿酒酵母中克隆覆盖整个Toledo基因组的16个重叠片段。然后，我们通过TAR逐步组装这些片段，直到整个基因组在酵母中重建。由于下一代序列分析显示低传代次数分离株代表亲本和成纤维细胞适应基因组的混合物，因此我们选择性地修饰成纤维细胞适应Toledo（Toledo-F）的单个DNA片段，并再次使用TAR组装来重建亲本Toledo（Toledo-P）。将线性全长HCMV基因组转染到人成纤维细胞中以回收病毒。与Toledo-F不同，Toledo-P表现出原代分离株的特征，包括体外广泛的细胞嗜性和在人源化小鼠中建立潜伏期和再激活的能力。因此，我们的新策略能够从头克隆CMV基因组，更有效的全基因组工程，并产生部分或完全来自合成DNA的病毒基因组。重要性大DNA病毒的基因组，如人巨细胞病毒（HCMV），很难使用目前的遗传工具进行操作，目前，不进行广泛的组织培养，不可能获得CMV的分子克隆。为了克服这些限制，我们使用合成生物学工具从病毒DNA中捕获基因组片段，并在酵母中组装全长基因组。使用含有野生型和组织培养适应病毒的混合物的HCMV分离株Toledo的早期传代。我们直接克隆了多数序列，并通过同时修饰多个基因组区域来重建少数序列。因此，我们的新方法提供了一种范例，不仅可以在全基因组范围内有效地工程化HCMV和其他大型DNA病毒，还可以促进原代分离株的克隆和遗传操作，并提供了一种生成完全合成基因组的途径。
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. 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.