Development of GenomeBuild as a Universal Method to Synthesize Genomes

GenomeBuild 的开发作为合成基因组的通用方法

• 批准号: 10565058
• 负责人: Mary Szatkowski Ozers
• 金额: $ 37.68万
• 依托单位: PROTEOVISTA, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-06 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10565058
• 关键词: Area; Bacteria; Bacterial Genome; Bar Codes; Base Pair Mismatch; Cell Line; Cells; Chromosome 22; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complementary DNA; Complex; Cost Savings; Custom; DNA; DNA Microarray Chip; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Devices; Environmental Science; Equation; Exhibits; FDA approved; Funding; Genes; Genetic; Genetic Polymorphism; Genome; Genomics; Goals; Grant; HCT116 Cells; Herpesvirus 1; Human Chromosomes; Human Genetics; Hybrids; Implant; Laboratories; Learning; Length; Libraries; Mammalian Chromosomes; Marketing; Medicine; Methodology; Methods; Mutation; Mycoplasma mycoides; Names; Nucleic Acids; Oligonucleotides; Oncolytic viruses; Organism; Peptide Nucleic Acids; Phase; Population; Process; Production; Protocols documentation; Provider; Reporting; Research; Research Personnel; Role; S phase; Saccharomyces cerevisiae; Savings; Site; Solid; Source; Synthesis Chemistry; Techniques; Technology; Television; Temperature; Therapeutic; Time; Viral Genome; Virus; Whole Organism; Work; Yeasts; arm; base; colon cancer cell line; commercialization; cost; cost effective; density; design; design,build,test; digital; ds-DNA; genome editing; genome-wide; human genomics; improved; insertion/deletion mutation; melanoma; melting; monomer; novel; prevent; restriction enzyme; scale up; screening; small molecule; solid state; success; synthetic biology; therapeutic development; whole genome

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.

项目摘要 基因组合成通用方法GenomeBuild的发展 商机编号：RFA-HG-20-016 PIS：克里斯托弗·L·沃伦和玛丽·S·奥泽斯 合成生物学包括从头合成大分子寡核苷酸并将其组装成 整个生物体的基因组，在医学、治疗学、环境等领域有着广泛的应用 科学和人类基因组学。然而，实现合成生物学全部潜力的能力一直是 受制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，没有经济高效的方法来引入广泛的基因编辑 变成基因组。“设计-建造-测试-学习-重复”的方法是合成生物学的基础，但 不能快速或经济有效地“构建”会抑制我们“学习”的能力，更不用说“重复”了。此外， 在负担得起的情况下，这一过程中可以包括各种不同的研究人员，而不仅仅是最 资助实验室和公司，以推动该领域向前发展并最大限度地提高其收益。定制的能力- 设计整个基因组不能用目前的基因组编辑技术，如CRISPR，和Solid- 基因组规模上的国家合成方法过于昂贵。在这份提案中，我们详细介绍了一种新的方法， GenomeBuild，以廉价、快速地合成高保真且完全可定制的病毒 从标准的DNA微阵列中提取基因组。作为原则证明，我们将合成150的修改形式 Kb HSV-1基因组，复制在TVEC(TVEC)中设计的遗传改变。 TVEC以免疫™的品牌上市，是美国食品和药物管理局批准的第一种用于 晚期黑色素瘤的治疗。尽管DNA微阵列可以作为一种直接且廉价的来源 一个复杂的寡核苷酸文库，它们相对于固相合成的高错误率排除了它们 在合成生物学中的有效应用。我们的技术将通过利用史无前例的 高密度肽核酸(PNA)阵列，用于去除从 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75kb的错误少于一个 平均序列。最后，这种定制的基因组将被引入使用标准方案的细胞系 并与基因上相同的非合成病毒进行感染性评估。便宜的 我们的GenomeBuild平台的成本和快速周转时间将使&gt；100kb合成 与商业上可用的产品相比，可以节省大量的时间和成本。vt.在.的基础上 成功开发后，GenomeBuild技术可以从这些努力推断为其他 基因组大小较大的生物，如其他病毒和细菌，使整个合成生物学 基因组和人类染色体，对任何实验室来说都更容易获得。