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: 10170542
• 负责人: Andrew D Ellington
• 金额: $ 18.3万
• 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: 2020-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170542
• 关键词: 2019-nCoV; Bacillus stearothermophilus; Biological Assay; Cells; Chimera organism; Chromosomes; Closure by clamp; Communities; Country; DNA; DNA Binding Domain; DNA Sequence; DNA amplification; DNA sequencing; DNA-Directed DNA Polymerase; Development; Directed Molecular Evolution; Emulsions; Engineering; Ensure; Enzymes; Genes; Genome; Goals; High temperature of physical object; In Vitro; Industry; Infrastructure; International; Introns; Kinetics; Lead; Length; Libraries; Medicine; Methods; Motion; Mutation; Organism; Patients; Performance; Polymerase; Preparation; Production; Promega; Property; Protein Engineering; RNA Splicing; RNA-Directed DNA Polymerase; Reagent; Research; Resources; Reverse Transcription; Saline; Sampling; Specificity; Speed; Stretching; Structure; System; Technology; Testing; Variant; Viral; Virus; Work; Writing; Yeasts; commercialization; grasp; high throughput screening; improved; molecular diagnostics; next generation; novel; novel sequencing technology; pandemic disease; point of care; sample collection; single cell sequencing; single molecule; synthetic biology; thermostability; tool; trizol; viral RNA; whole genome

Supplemental Project Summary (derived from the original, changes underlined) 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 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. The methods developed in the execution of this proposal should also prove extremely useful for greatly improved reagents for molecular diagnostics for SARS-CoV-2. 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). Using the methods in Aim 1.2, we can produce polymerase variants that should be able to directly participate in RT-qPCR without sample preparation, including from samples inactivated with denaturants. 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. Using the methods described in Aim 3.1, we can generate thermostable reverse transcriptases that should prove useful for the development of isothermal amplification assays that can be used at point-of-care, or in resource-poor settings. 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. In the current crisis, polymerase engineering for particular functions, directed towards needs that the community has and that need to be resolved for forward motion on testing, is a critical component of a national plan.

补充项目摘要(源自原文，有下划线的更改) 合成生物学的进步已经加速到现在整个基因组的合成 有可能。然而，这些壮举的技术是艰苦的，并且产生了新的染色体 或者基因组需要多年的努力，从小片段到更大的组装。速度 如果有可能常规地进行大片段组装，大片段组装的(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(&gt；100kb)。在执行本建议过程中制定的方法 对于大大改进SARS-CoV-2的分子诊断试剂，也应该被证明是极其有用的。至 为此，这项建议的重点是进一步发展一种新的定向进化方法，称为 隔室自复制(CSR)，在这种情况下，乳剂中细胞中表达的聚合酶经历 热循环来放大他们自己的基因，产生应该被证明是有能力的长阅读DNA聚合酶 产生长度为100kb的聚合酶链式反应扩增片段，几乎没有错误。为了达到这一目标，我们建议制定一项 一种新的文库构建方法，该方法最有效地将序列和结构域从 各种DNA聚合酶变体以形成不同的嵌合体(目标1.1)，并使用以下方法筛选这些文库 对CSR的改进，使我们能够在酵母中选择极高的加工性(目标1.2)和有效的错误- 更正(目标1.3)。使用AIM 1.2中的方法，我们可以产生应该能够 直接参与RT-qPCR，不需要样品准备，包括使用变性剂灭活的样品。 结果将以它们在体外合成长扩增子的能力为特征(目标2.1)，用于 它们的保真度(目标2.2)，以及它们的详细动力学性质(目标2.3)。最后，为了更好地保证过程性 在得到的聚合酶嵌合体中，我们将添加DNA结合域(AIM 3.1)或夹子(AIM 3.2)这应该会带来更好的掌握DNA的能力。使用目标3.1中描述的方法，我们可以生成 耐热逆转录酶应被证明对等温扩增的发展有用 可在护理地点或在资源匮乏的环境中使用的检测。除了加速正在进行的 基因组合成的革命，这种长时间阅读的聚合酶也应该为新的测序铺平道路 技术，包括单分子测序和单细胞测序。在当前的危机中， 针对特定功能的聚合酶工程，针对社区拥有和需要的需求 为推进试验而解决的问题，是国家计划的关键组成部分。