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：
David Craford
金额：
$ 101.94万
依托单位：
BACSTITCH DNA INC.
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

来源：
https://reporter.nih.gov/project-details/10760876
关键词：
Acceleration Agar Bacteria Bar Codes Biotechnology Businesses Cloning Complex Custom DNA DNA Sequence DNA Structure DNA biosynthesis Data Set Databases Enzymes Future Genes Genetic Genetic Conjugation Genomics Goals Government Grant Guanine + Cytosine Composition In Vitro Industrialization Libraries Licensing Marketing Methods Oligonucleotides Output Partner in relationship Pathway interactions Pharmacologic Substance Phase Plasmids Polymerase Positioning Attribute Preparation Printing Procedures Production Provider Reagent Research Robotics Sequence Analysis Services Small Business Innovation Research Grant Standardization System Systems Analysis Technology Time Validation analysis pipeline antibody engineering base cellular engineering combinatorial commercialization cost design,build,test drug development flexibility frontier in vivo large scale production meetings metabolic engineering new technology novel programs research and development synthetic biology

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.

项目概要/摘要通过促进以前无法获得的基因和遗传途径的生产和表征，长而复杂的 DNA 的高通量、低成本合成可以开辟合成生物学、药物开发和基因组学的新领域。微阵列打印技术可以以高通量和低成本合成几乎任何长达 300 个碱基的寡核苷酸，而酶合成等新技术则有可能从头合成长达 2kb 的序列。为了构建 2-5kb DNA 块，可以使用一套体外技术来组装寡核苷酸，包括聚合酶循环组装、Gibson 组装和 Golden Gate 组装。然而，应用体外技术组装长于约 2-5kb 的 DNA 需要大量的实践时间，难以多重化和规模化，对于复杂 DNA 的构建不可靠（例如极端 GC 含量、同聚物、重复序列、DNA 结构），并且对于长度超过 ~7kb 的 DNA 来说极具挑战性。此外，目前筛选序列完美克隆的组装方法非常昂贵，并且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，我们开发了一种新颖、低成本、高度多重的体内方法来组装长且复杂的 DNA，并对克隆进行测序验证。在这种方法中，DNA块阵列被引入质粒和细菌中，这些阵列通过细菌交配顺序缝合在一起。组装后，细菌阵列通过另一轮细菌交配进行条形码标记，从而能够从合并的克隆中进行大规模并行 DNA 分离和测序文库制备。与现有的体外克隆相比，我们将寡核苷酸或 DNA 块拼接在一起的方法比当前技术便宜 100 倍，所需的动手时间减少 100 倍，而总时间没有增加。重要的是，体内组装可以大规模组装至少 15kb 且包含许多复杂 DNA 区域的 DNA。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，以及 ii) 构建有助于跟踪和预测的序列分析管道和数据库来强化、扩展和商业化这项技术。体内 DNA 组装的错误模式。这些目标将使 BacStitch 能够首先提供定制服务，并最终提供标准化产品，以满足对长而复杂 DNA 的未满足需求。