Multiplexed in vivo assembly of long and complex DNA

长且复杂的 DNA 的多重体内组装

基本信息

  • 批准号:
    10760876
  • 负责人:
  • 金额:
    $ 101.94万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2023
  • 资助国家:
    美国
  • 起止时间:
    2023-07-10 至 2025-06-30
  • 项目状态:
    未结题

项目摘要

PROJECT SUMMARY/ ABSTRACT High-throughput, low-cost synthesis of long and complex DNA could open up new frontiers in synthetic biology, drug development, and genomics by facilitating the production and characterization of previously inaccessible genes and genetic pathways. Microarray printing technologies provide for high-throughput and low-cost synthesis of almost any oligonucleotide up to 300 bases long, while new technologies, such as enzymatic synthesis, have the potential to synthesize sequences de novo up to 2kb. To construct 2-5kb DNA blocks, oligonucleotides can be assembled using a suite of in vitro technologies, including polymerase cycling assembly, Gibson assembly, and Golden Gate assembly. However, application of in vitro technologies to assemble DNA longer than ~2-5kb requires considerable hands-on time, is difficult to multiplex and scale, is unreliable for construction of complex DNA (e.g. extreme GC content, homopolymers, repeats, DNA structure), and is extremely challenging for DNA longer than ~7kb. In addition, current methods to screen assemblies for sequence perfect clones are expensive and require considerable hands-on time or complex robotics. To overcome these hurdles, we have developed a novel, low-cost, highly multiplexed in vivo method to assemble long and complex DNA, and to sequence verify clones. In this method, arrays of DNA blocks are introduced into plasmids and bacteria, and these arrays are stitched together sequentially by bacterial mating. Following assembly, bacterial arrays are barcoded via another round of bacterial mating, enabling massively parallel DNA isolation and sequencing library preps from pooled clones. Compared to existing in vitro cloning our approach for stitching either oligos or DNA blocks together is up to 1 00X cheaper and requires up to 100X less hands-on time than current technologies, with no increase in total time. Importantly, in vivo assembly can assemble, at scale, DNA that is at least 15kb and that contains many regions of complex DNA. Here, we propose to harden, scale, and commercialize this technology by i) building a high-throughput production pipeline for in vivo DNA assembly and sequence validation, and ii) building a sequence analysis pipeline and database that will aid in both tracking and predicting error modes of in vivo DNA assembly. These aims will enable BacStitch to initially provide custom services and eventually standardized products that fulfill an unmet need for long and complex DNA.
项目摘要/摘要 长而复杂的DNA的高通量,低成本合成可以通过促进先前不可接近的基因和遗传途径的产生和表征来打开合成生物学,药物发育和基因组学的新领域。微阵列打印技术可为几乎所有最高300个碱基长的寡核苷酸的高通量和低成本合成,而诸如酶促合成之类的新技术具有合成序列的潜力,最高可达2KB。为了构建2-5KB DNA块,可以使用一套体外技术组装寡核苷酸,包括聚合酶循环组件,Gibson组装和Golden Gate组装。然而,在〜2-5kb的组装中,应用体外技术需要相当大的动手时间,难以多重多重,并且对复杂DNA的构建(例如,极端的GC含量,均聚物,重复,DNA结构,DNA结构)是不可靠的,并且对于DNA的DNA比〜7KB长。此外,当前筛选序列完美克隆组件的方法很昂贵,需要大量的动手时间或复杂的机器人技术。为了克服这些障碍,我们开发了一种新型的低成本,高度多重的体内方法来组装长而复杂的DNA,并为验证克隆进行测序。在这种方法中,将DNA块的阵列引入质粒和细菌中,这些阵列通过细菌交配顺序缝合在一起。组装后,通过另一轮细菌交配对细菌阵列进行条形码,从而实现了大量平行的DNA分离,并从合并的克隆中准备了测序文库。与现有的体外克隆我们将寡核体或DNA块拼接的方法相比,最高可便宜1 00倍,并且比目前的技术需要少100倍的动手时间,而总时间没有增加。重要的是,体内组装可以按至少15kb的DNA组装,其中包含许多复杂DNA区域。在这里,我们建议通过i)构建用于体内DNA组装和序列验证的高通量生产管道来加强,扩展和商业化这项技术,ii)构建序列分析管道和数据库,该管道和数据库将有助于跟踪和预测体内DNA组装中的误差模式。这些目标将使Bactitch最初提供自定义服务,并最终提供对长而复杂的DNA需求的标准化产品。

项目成果

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