Rapid and efficient generation of sequence variants by templated synthesis

项目摘要 合成基因和通路大小的DNA的能力是功能基因组学中的关键工具。许多应用 基因合成需要产生和测试含有多位点突变的序列变体。因为- 然而，目前生产这种构建体的方法仍然依赖于从头合成，这仍然缓慢，昂贵， 并且对于变体生成不匹配。在这里，我们提出了模板引导基因合成的概念， 论文作为一种新的策略，以建立内切酶大小的DNA结构使用现有的序列作为模板。我们 第一个目标将是通过开发预测性的分子标记来推进这种模板指导的合成和诱变方法。 寡核苷酸设计算法，优化实验方案，实现机器人自动化。我们的第二 AIM将专注于通过基于珠的寡核苷酸捕获技术将该方法扩展到每次> 1，000个构建体 以及皮升乳液液滴中的平行变体组件。我们将评估我们平台的性能 在一个示例用例中，通过产生磷酸模拟变体来映射细胞信号传导。我们预计 我们的模板引导合成平台将能够产生>5 kb的多位点序列变体 以高通量和自动化的方式在长度上进行。此外，这种方法将高达10-20倍 比目前的从头基因合成方法更便宜和快5-10倍。

PROJECT SUMMARY The ability to synthesize gene- and pathway-sized DNA is a key tool in functional genomics. Many applications of gene synthesis call for the generation and testing of sequence variants containing multi-site mutations. Unfor- tunately, current approach to produce such constructs still rely on de novo synthesis, which remains slow, costly, and ill-matched for variant generation. Here, we propose to advance the concept of template-guided gene syn- thesis as a novel strategy to build kilobase-sized DNA constructs using an existing sequence as a template. Our first Aim will be to advance this template-guided synthesis and mutagenesis approach by developing predictive oligo design algorithms, optimizing experimental protocols, and implementing robotic automation. Our second Aim will focus on extending the method to >1,000 constructs at a time with bead-based oligo capture techniques and parallel variant assemblies in picoliter emulsion droplets. We will evaluate the performance of our platform in an example use case through the generation of phosphomimetic variants to map cell signaling. We expect that our template-guided synthesis platform will be able to produce multi-site sequence variants that are >5 kb in length in a high-throughput and automated fashion. Furthermore, this approach will be up to 10-20 times cheaper and 5-10 time faster than current de novo gene synthesis methods.