Directed evolution of polymerases that can read and write extremely long sequences

聚合酶的定向进化可以读取和写入极长的序列

• 批准号: 10548111
• 负责人: Andrew D Ellington
• 金额: $ 35.08万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10548111
• 关键词: Acceleration; Cells; Chimera organism; Chromosomes; Closure by clamp; Collaborations; Communities; DNA; DNA Binding Domain; DNA Library; DNA Sequence; DNA amplification; DNA sequencing; DNA-Directed DNA Polymerase; Development; Directed Molecular Evolution; Elements; Emulsions; Ensure; Enzymes; Feedback; Genes; Genome; Goals; In Vitro; Individual; Industry; Kinetics; Length; Libraries; Measures; Medicine; Methods; Mutation; Organism; Polymerase; Polymerase Gene; Process; Production; Property; Reading; Reagent; Research; Series; Speed; Stretching; Technology; Tertiary Protein Structure; Variant; Writing; Yeasts; design; design and construction; follow-up; genetic variant; grasp; homologous recombination; improved; method development; next generation; next generation sequencing; novel; novel sequencing technology; rational design; single cell sequencing; single molecule; synthetic biology; tool; whole genome

Project Summary Advances in synthetic biology have accelerated to the point where the synthesis of entire genomes is now possible. However, the technologies for these feats are painstaking, and the production of a new chromosome or genome requires multiple years of effort, working from small fragments to ever larger assemblies. The cumbersome assembly process is due in large measure to the need to carry out an ordered series of hierarchical homologous recombination steps that proceed through transformations into organisms, primarily yeast. The speed (and ultimately scale) of large fragment assembly would be greatly improved if it were possible to routinely amplify very long stretches of DNA (> 100 kb) in vitro. To that end, this proposal is focused on the further development of a novel directed evolution method known as Compartmentalized Self-Replication (CSR), in which polymerases expressed in cells in emulsions undergo thermal cycling to amplify their own genes, to generate long read DNA polymerases that should prove capable of generating PCR amplicons > 100 kb in length, with few errors. To achieve this goal, we propose to develop a novel library construction method that most efficiently brings together sequence and structural domains from a variety of DNA polymerase variants to form diverse chimeras (Aim 1.1), and to sieve these libraries using improvements to CSR that will allow us to select for extreme processivity in yeast (Aim 1.2) and efficient error-correction (Aim 1.3). The variants that result will be characterized for their ability to synthesize long amplicons in vitro (Aim 2.1), for their fidelity (Aim 2.2), and for their detailed kinetic properties (Aim 2.3). Finally, to better ensure the processivity of the resultant polymerase chimeras, we will append either DNA-binding domains (Aim 3.1) or clamps (Aim 3.2) that should lead to much better ability to grip DNA. In addition to accelerating the ongoing revolution in genome synthesis, such long-read polymerases should also pave the way to new sequencing technologies, including for single molecule sequencing and for single cell sequencing.

项目概要 合成生物学的进步已经加速到现在可以合成整个基因组 可能的。然而，实现这些壮举的技术是艰苦的，并且新染色体的产生 或基因组需要多年的努力，从小片段到更大的组装体。这 繁琐的组装过程在很大程度上是由于需要进行一系列有序的分层组装 通过转化为生物体（主要是酵母）进行的同源重组步骤。这 如果能够常规地进行大碎片组装的速度（以及最终的规模）将会大大提高 在体外扩增非常长的 DNA 片段 (> 100 kb)。为此，本提案的重点是进一步 开发了一种称为分区自我复制（CSR）的新型定向进化方法 在乳液中的细胞中表达的聚合酶经历热循环以扩增它们自己的基因，以 生成长读长 DNA 聚合酶，应证明能够生成长度 > 100 kb 的 PCR 扩增子， 几乎没有错误。为了实现这一目标，我们建议开发一种最新颖的图书馆构建方法 有效地将来自多种 DNA 聚合酶变体的序列和结构域聚集在一起，形成 不同的嵌合体（目标 1.1），并使用 CSR 的改进来筛选这些库，这将使我们能够选择 用于酵母的极端持续性（目标 1.2）和有效的纠错（目标 1.3）。产生的变体将 表征其体外合成长扩增子的能力（目标 2.1）、保真度（目标 2.2），以及 了解其详细的动力学特性（目标 2.3）。最后，为了更好地保证所得聚合酶的持续合成能力 嵌合体，我们将附加 DNA 结合域（目标 3.1）或夹子（目标 3.2），这应该会导致很多 更好的抓住 DNA 的能力。除了加速正在进行的基因组合成革命之外，这种长读 聚合酶还应该为新的测序技术铺平道路，包括单分子测序技术 测序和单细胞测序。