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

项目摘要 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。的 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 同源重组步骤，通过转化到生物体中进行，主要是酵母。的 如果可以常规地 体外扩增非常长的DNA片段（> 100 kb）。为此目的，本建议的重点是进一步 开发一种新的定向进化方法，称为区室化自我复制（CSR）， 所述聚合酶在乳液中的细胞中表达，经历热循环以扩增它们自身的基因， 产生长读段DNA聚合酶，其应证明能够产生长度> 100 kb的PCR扩增子， 几乎没有错误。为了实现这一目标，我们建议开发一种新的库构建方法， 有效地将来自各种DNA聚合酶变体的序列和结构域结合在一起， 多样的嵌合体（目标1.1），并使用CSR的改进筛选这些文库，这将使我们能够选择 用于酵母中的极端持续合成能力（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），其保真度（目标2.2），和 详细的动力学性质（目标2.3）。最后，为了更好地确保所得聚合酶的持续合成能力， 在嵌合体中，我们将添加DNA结合结构域（Aim 3.1）或夹子（Aim 3.2），这将导致更多的 更好地抓住DNA。除了加速正在进行的基因组合成革命之外，这种长期的阅读 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。

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.