Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides.

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 system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, 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, 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 and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the 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.