Development of GenomeBuild as a Universal Method to Synthesize Genomes

项目摘要 GenomeBuild作为通用基因组合成方法的发展 机会编号：RFA-HG-20-016 PI：Christopher L.沃伦和玛丽S.厄泽尔 合成生物学包括从头合成和组装大的寡核苷酸成寡核苷酸的能力。 整个生物体的基因组，在医学、治疗学、环境保护、 科学和人类基因组学。然而，实现合成生物学的全部潜力的能力一直是 由于构建合成基因组和大型哺乳动物染色体的时间和成本限制， 使用当前的技术。此外，没有成本有效的方法来引入广泛的基因编辑 转化成基因组“设计-构建-测试-学习-重复”方法是合成生物学的基础， 无法快速或经济有效地“构建”抑制了我们“学习”的能力，更不用说“重复”了。此外，委员会认为， 由于负担得起，各种各样的研究人员都可以参与这一过程，而不仅仅是最高级别的研究人员。 资助的实验室和公司，以推动该领域的发展，并最大限度地提高其收益。定制的能力- 设计一个完整的基因组不能用目前的基因组编辑技术，如CRISPR，和固体- 在基因组规模上的状态合成方法太昂贵。在这个建议中，我们详细介绍了一种新的方法， GenomeBuild，以廉价和快速合成高保真和完全可定制的病毒 从标准的DNA微阵列中提取基因组。作为原理证明，我们将合成150的修改形式， kb HSV-1基因组，其复制在talimogene laherparepvec（TVEC）中设计的遗传改变。 TVEC，以商标名Imlygic™销售，是第一个FDA批准的用于肿瘤的溶瘤病毒。 治疗晚期黑色素瘤虽然DNA微阵列可以作为一个直接和廉价的来源， 寡核苷酸的复杂文库，它们相对于固相合成的高错误率已经排除了它们的 在合成生物学中的有效应用我们的技术将通过利用前所未有的 高密度肽核酸（PNA）阵列，以去除从 相应的DNA微阵列。我们的目标是合成高保真的微阵列提取的基因组 可以组装成修饰的病毒基因组的寡核苷酸，每75 kb的寡核苷酸具有小于一个错误， 序列平均。最后，将使用标准方案将这种定制基因组引入细胞系中 并与遗传上等同的非合成病毒相比评估感染性。廉价的 我们的GenomeBuild平台的成本和快速周转时间将允许生产>100 kb的合成 与商业上可获得的产品相比，可以显著节省时间和成本。后 成功的开发，基因组构建技术可以从这些努力中推断出更多的 生物体，如其他病毒和细菌与较大的基因组大小，使合成生物学的整体 基因组和人类染色体，任何实验室都更容易获得。

Project Summary Development of GenomeBuild as a Universal Method to Synthesize Genomes Opportunity Number: RFA-HG-20-016 PIs: Christopher L. Warren and Mary S. Ozers Synthetic biology encompasses the ability to de novo synthesize and assemble large oligonucleotides into genomes of whole organisms, with a broad range of applications in medicine, therapeutics, environmental sciences, and human genomics. However, the ability to realize the full potential of synthetic biology has been hampered by the time and cost limitations of building synthetic genomes and large mammalian chromosomes using current techniques. Additionally, there is no cost effective method to introduce extensive genetic edits into genomes. The “Design-Build-Test-Learn-Repeat” approach is fundamental to synthetic biology but the inability to “Build” quickly or cost-effectively inhibits our ability to “Learn,” much less “Repeat.” Furthermore, with affordability, a diverse array of researchers can be included in this process, not only the most highly funded laboratories and companies, to propel the field forward and maximize its gains. The ability to custom- design an entire genome cannot be done with current genomic editing techniques such as CRISPR, and solid- state synthesis methods on the genome scale are too expensive. In this proposal, we detail a novel method, GenomeBuild, to inexpensively and rapidly synthesize a high fidelity and completely customizable viral genome from a standard DNA microarray. As proof-of-principle, we will synthesize a modified form of the 150 kb HSV-1 genome that reproduces the genetic alterations designed in talimogene laherparepvec (TVEC). TVEC, marketed under the brand name Imlygic™, is the first FDA approved oncolytic virus used for the treatment of advanced melanoma. Although DNA microarrays can serve as a direct and inexpensive source for a complex library of oligonucleotides, their high error-rate relative to solid phase synthesis have precluded their effective use in synthetic biology. Our technology will circumvent this problem by harnessing an unprecedented high-density peptide nucleic acid (PNA) array to remove imperfect DNA sequences obtained from a corresponding DNA microarray. Our aims will synthesize high-fidelity microarray-extracted genomic oligonucleotides that can be assembled into a modified viral genome with less than one error per 75 kb of sequence on average. Finally, this custom genome will be introduced into a cell line using standard protocols and assessed for infectivity as compared to a genetically equivalent non-synthesized virus. The inexpensive cost and fast turnaround time of our GenomeBuild platform will allow production of >100 kb synthetic oligonucleotides at a significant time and cost savings compared to commercially available products. Upon successful development, the GenomeBuild technology can be extrapolated from these efforts to additional organisms such as other viruses and bacteria with larger genomic sizes, making synthetic biology of whole genomes, and human chromosomes, more attainable for any laboratory.