Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

中文翻译：

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使用低成本序列验证的阵列合成寡核苷酸合成无细胞噬菌体基因组。

合成用于人类的工程噬菌体（噬菌体）在从使用噬菌体展示的药物筛选到使用噬菌体疗法的临床应用等各种应用中具有潜力。然而，噬菌体的工程改造通常涉及使用体内系统，该系统由于对宿主的高毒力和低转化效率而具有低生产效率。为了避免这些问题，使用化学合成的寡核苷酸（寡核苷酸）从头进行噬菌体基因组合成增加了在无细胞系统中工程化噬菌体的潜力。在这里，我们介绍了无电池，低成本，从头开始基因合成技术称为Sniper组装，用于噬菌体基因组构建。通过对芯片合成的寡核苷酸进行大规模并行测序，我们在体外产生并鉴定了约100 000个克隆DNA簇，在无细胞环境中鉴定了5000个无错误的簇。为了证明其实际应用，我们使用65个经过序列验证的DNA克隆合成了不动杆菌噬菌体AP205基因组（4268 bp）。与以前的报告相比，Sniper组装通过生产低成本的序列验证DNA降低了基因组合成成本（$ 0.0137 / bp）。