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 开发基因组建筑作为综合基因组的通用方法 机会编号：RFA-HG-20-016 PIS：Christopher L. Warren和Mary S. Ozers 合成生物学包括从头合成并组装大寡核苷酸的能力 整个生物体的基因组，在医学，治疗，环境中具有广泛的应用 科学和人类基因组学。但是，实现合成生物学的全部潜力的能力已经 受到建筑合成基因组和大型哺乳动物染色体的时间和成本限制的阻碍 使用当前技术。此外，没有具有成本效益的方法来引入广泛的遗传编辑 进入基因组。 “设计建造测试 - 与重复”方法是合成生物学的基础 无法快速或成本效率地“构建”会抑制我们“学习”的能力，更少的“重复”。此外， 有了可用性，可能包括一系列研究人员，不仅是最高的 资助实验室和公司，以推动该领域的前进并最大化其收益。定制的能力 - 设计当前的基因组编辑技术（例如CRISPR和固体）无法完成整个基因组 基因组量表上的状态合成方法太昂贵了。在此提案中，我们详细介绍了一种新颖的方法 基因组建筑，廉价，迅速地合成高保真度和完全可定制的病毒 来自标准DNA微阵列的基因组。作为原理证明，我们将合成150的修改形式 KB HSV-1基因组，该基因组重现了塔利莫烯laherparepvec（TVEC）中设计的遗传改变。 TVEC以品牌名称Imlygic™销售，是FDA批准的溶瘤病毒 尽管DNA微阵列可以作为直接且廉价的来源 一个复杂的寡核苷酸库，它们相对于固相合成的高误差已排除 有效地用于合成生物学。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A的不完善的DNA序列 相应的DNA微阵列。我们的目标将综合高保真微阵列提取的基因组 可以组装成修饰的病毒基因组中的寡核苷酸，每75 kb少于一个误差 序列平均。最后，该自定义基因组将使用标准协议引入细胞系 与遗传等效的非合成病毒相比，评估了感染。便宜的 我们的基因组建平台的成本和快速周转时间将允许生产> 100 kb的合成时间 与市售产品相比，寡核苷酸在很大的时间和成本节省。之上 成功开发，基因组建技术可以从这些努力中推断出来 诸如其他病毒和具有较大基因组大小的细菌之类的生物，使整个生物学的合成生物学 基因组和人类染色体，对于任何实验室都可以实现。