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（源自原始文件，更改部分加下划线） 合成生物学的进步已经加速到现在整个基因组的合成 可能然而，这些壮举的技术是艰苦的，新染色体的生产 或者基因组需要多年的努力，从小片段到更大的组合。速度 (and如果有可能常规地 体外扩增非常长的DNA片段（> 100 kb）。在执行本提案过程中制定的方法 也应该证明是非常有用的，大大改善了试剂的分子诊断SARS-CoV-2。到 为此，该建议的重点是进一步发展一种新的定向进化方法，称为 区室化自我复制（CSR），其中乳液中细胞中表达的聚合酶经历 热循环来扩增它们自己的基因，以产生长读段DNA聚合酶， 产生长度> 100 kb的PCR扩增子，几乎没有错误。为达致这个目标，我们建议制定一套 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 多种DNA聚合酶变体以形成多种嵌合体（Aim 1.1），并使用 对CSR的改进，使我们能够选择极端的酵母持续合成能力（目标1.2）和有效的错误- 校正（目标1.3）。使用目标1.2中的方法，我们可以生产聚合酶变体，这些变体应该能够 直接参与RT-qPCR而无需样品制备，包括来自用变性剂灭活的样品。 将表征所产生的变体在体外合成长扩增子的能力（目的2.1）， 它们的保真度（目标2.2），以及它们详细的动力学性质（目标2.3）。最后，为了更好地保证 在所得到的聚合酶嵌合体中，我们将附加DNA结合结构域（Aim 3.1）或夹子（Aim 3.2)这应该会导致更好的抓持DNA的能力。使用目标3.1中描述的方法，我们可以生成 热稳定的逆转录酶，应该证明对等温扩增的发展有用 可以在护理点或资源贫乏的环境中使用的测定。除了加快正在进行的 基因组合成的革命，这种长读段聚合酶也应该为新的测序铺平道路 技术，包括用于单分子测序和用于单细胞测序。在当前的危机中， 针对特定功能的聚合酶工程，针对社区的需要和需要 这是国家计划的一个关键组成部分。