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（源自原始的，带下划线的更改） 合成生物学的进步已加速到现在的整个基因组的合成 可能的。但是，这些壮举的技术是艰苦的，以及新染色体的生产 或基因组需要多年的努力，从小碎片到较大的组合。速度 （并最终缩放）大型碎片组装将大大改善，如果有可能进行常规 在体外扩增非常长的DNA（> 100 kb）。在执行本提案中开发的方法 还应该证明，对于SARS-COV-2的分子诊断试剂，应非常有用。到 这一最终，该提案的重点是进一步发展一种新型的定向进化方法，称为 分隔自我复制（CSR），其中乳液中细胞中表达的聚合酶经历 热循环以扩大自己的基因，生成长读取DNA聚合酶，应证明能够 生成PCR扩增子的长度> 100 kb，几乎没有错误。为了实现这一目标，我们建议开发 新的图书馆构造方法最有效地从一个 多种DNA聚合酶变体形成各种嵌合体（AIM 1.1），并使用这些文库筛选这些库 对CSR的改进，这将使我们能够在酵母（AIM 1.2）和有效错误中选择极端加工性 - 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的聚合酶变体 直接参与RT-QPCR，而无需样品制备，包括从变性剂灭活的样品中。 结果的变体以其体外合成长扩增子（AIM 2.1）的能力而被特征 他们的保真度（AIM 2.2）及其详细的动力学特性（AIM 2.3）。最后，更好地确保过程 在所得的聚合酶嵌合体中，我们将附加DNA结合域（AIM 3.1）或夹具（AIM 3.2）这应该提高抓地力DNA的能力得多。使用AIM 3.1中描述的方法，我们可以生成 热稳定的逆转录酶，应证明对等温扩增有用 可以在护理点或资源贫乏的设置中使用的测定。除了加速持续的 基因组综合革命，这种长阅读聚合酶也应该为新测序铺平道路 技术，包括用于单分子测序和单细胞测序。在当前的危机中 用于特定功能的聚合酶工程，针对社区的需求和需求 要解决测试的远期运动，这是国家计划的关键组成部分。