Multiplexed In Vivo DNA Assembly

多重体内 DNA 组装

• 批准号: 10620139
• 负责人: SASHA F LEVY
• 金额: $ 53.59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-10 至 2023-10-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10620139
• 关键词: Bacteria; Bacterial Artificial Chromosomes; Biomedical Research; Cells; Cloning; Complex; DNA; DNA Library; DNA biosynthesis; Detection; Elements; Escherichia coli; Generations; Genes; Genetic; Genome; Genomics; Goals; In Vitro; Individual; Learning; Length; Libraries; Liquid substance; Methods; Modality; Oligonucleotides; Open Reading Frames; Operon; Output; Partner in relationship; Pathway interactions; Phase; Plasmids; Polymerase; Positioning Attribute; Printing; Procedures; Process; Production; Regulatory Element; Robot; Series; Services; System; Techniques; Technology; Testing; Time; Variant; Vendor; Work; Yeasts; base; cost; frontier; gene synthesis; in vivo; incremental cost; new technology; novel; synthetic biology; technology development

PROJECT SUMMARY/ABSTRACT High-throughput and low-cost synthesis of long DNA fragments could open up new frontiers in genomics and synthetic biology by facilitating the production of genes, genetic pathways, variant libraries, or entire synthetic genomes. Microarray printing technologies provide for high-throughput and low-cost synthesis of short oligonucleotides (currently up to 300 bases long), while new technologies, such as enzymatic synthesis, have the potential to synthesize longer sequences. Libraries of open reading frames or regulatory elements have also been constructed. However, assembly of the above or other DNA elements into longer fragments remains a challenge. Widely-used in vitro DNA assembly methods, such as Gibson assembly and polymerase chain assembly, require considerable hands-on time, are difficult to multiplex and scale, and have size constraints on the length of the assembled product. In addition, accurate long assemblies from de novo synthesized DNA are difficult to achieve without a high-throughput error correction step. To overcome these hurdles, we have developed a novel, multiplexed method to parse, sequence verify, and stitch together DNA of various sizes in vivo. Using this method, complex pools of array-synthesized oligonucleotides or other DNA elements can be turned into positionally-ordered cellular arrays, with each position containing a sequence-verified input DNA. Input DNA in these arrays can then be stitched together recursively by bacterial mating. A key feature of our technology is early error detection, whereby individual DNA inputs are sequence-verified prior to stitching. That is, errors that occur during short DNA synthesis are detected and removed, rather than propagated into longer assemblies. At maximal throughput, we estimate that our technology has the potential to produce thousands of DNA assemblies in parallel at a cost that is at least an order of magnitude cheaper than commercially available gene synthesis technologies. Because DNA stitching can occur within bacterial artificial chromosomes, in vivo DNA parsing and stitching can perform multiplexed assembly of DNA constructs exceeding 100kb, at least an order of magnitude longer than current in vitro assembly techniques. In vivo assembly uses a stepwise stitching process that can be branched, allowing the construction of a wide variety of variants at low incremental cost. Here, we propose to continue development of this technology i) by building many genes to learn the error modalities of the process, ii) by assembling a ~100kb product, and iii) by developing a multi-step assembly procedure to reduce assembly time and increase product accuracy. Together, this integrated set of aims will result in a new high-throughput DNA assembly platform capable of synthesizing long and complex DNA constructs and variant libraries at low cost.

项目总结/摘要 长DNA片段的高通量和低成本合成可以通过促进基因、遗传途径、变体文库或整个合成基因组的产生来开辟基因组学和合成生物学的新前沿。微阵列打印技术提供了短寡核苷酸（目前长达300个碱基）的高通量和低成本合成，而新技术，如酶促合成，具有合成更长序列的潜力。还构建了开放阅读框或调控元件的文库。然而，将上述或其他DNA元件组装成更长的片段仍然是一个挑战。广泛使用的体外DNA组装方法，如吉布森组装和聚合酶链组装，需要相当长的动手时间，难以多重化和规模化，并且对组装产物的长度具有尺寸限制。此外，在没有高通量纠错步骤的情况下，难以实现从从头合成的DNA的精确长组装。为了克服这些障碍，我们开发了一种新的，多路复用的方法来解析，序列验证，并在体内缝合各种大小的DNA。使用这种方法，阵列合成的寡核苷酸或其他DNA元件的复杂池可以变成位置有序的细胞阵列，每个位置包含序列验证的输入DNA。然后，这些阵列中的输入DNA可以通过细菌交配递归地缝合在一起。我们的技术的一个关键特征是早期错误检测，即在拼接之前对单个DNA输入进行序列验证。也就是说，在短DNA合成过程中发生的错误被检测和删除，而不是传播到更长的组装中。在最大通量下，我们估计我们的技术有可能以比市售基因合成技术至少便宜一个数量级的成本并行生产数千个DNA组装体。由于DNA拼接可以发生在细菌人工染色体内，体内DNA解析和拼接可以进行超过100 kb的DNA构建体的多重组装，比目前的体外组装技术长至少一个数量级。体内组装使用可以分支的逐步缝合过程，允许以低增量成本构建各种变体。在这里，我们建议继续开发这项技术，i）通过构建许多基因来学习过程的错误模式，ii）通过组装~ 100 kb的产品，iii）通过开发多步组装程序来减少组装时间并提高产品准确性。总之，这一整套目标将导致一个新的高通量DNA组装平台，能够以低成本合成长而复杂的DNA构建体和变体文库。