Authors and Affiliations

Authors and Affiliations

Namjin Cho^1,†, Han Na Seo^1,†, Taehoon Ryu^2,†, Euijin Kwon², Sunghoon Huh¹, Jinsung Noh³, Huiran Yeom³, Byungjin Hwang¹, Heejeong Ha¹, Ji Hyun Lee^4,5, Sunghoon Kwon^3,6,7,* and Duhee Bang<sup>1,*

1</sup>Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea, ²Celemics Inc., 371-17, Gasan-dong, Geumcheongu, Seoul 153-718, Republic of Korea, ³Department of Electrical and Computer Engineering, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea., ⁴Department of Clinical Pharmacology and Therapeutics, College of Medicine, Kyung Hee University, Seoul 02447, Republic of Korea, ⁵Kyung Hee Medical Science Research Institute, Kyung Hee University, Seoul 02447, Republic of Korea, ⁶ Institute of Entrepreneurial Bio Convergence, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. and ⁷Seoul National University Hospital Biomedical Research Institute, Seoul National University Hospital, 101, Daehak-ro Jongno-gu, Seoul, 03080, Republic of Korea.

^†These authors contributed equally to this work as first authors.

^*To whom correspondence should be addressed. 

Correspondence may also be addressed to Sunghoon Kown. 

Selective retrieval of sequence-verified oligonucleotides (oligos) from next-generation sequencing (NGS) flow cells, termed megacloning, promises accurate and reliable gene synthesis. However, gene assembly requires a complete collection of overlapping sense and nonsense oligos, and megacloning does not typically guarantee the complete production of sequence-verified oligos. Therefore, missing oligos must be provided via repetitive rounds of megacloning, which introduces a bottleneck for scaled-up efforts at gene assembly. Here, we introduce the concept of high-depth tiled oligo design to successfully utilize megacloned oligos for gene synthesis. Using acquired oligos from a single round of the megacloning process, we assembled 72 of 81 target Cas9-coding gene variants. We further validated 62 of these cas9 constructs, and deposited the plasmids to Addgene for subsequent functional characterization by the scientific community. This study demonstrates the utility of using sequence-verified oligos for DNA assembly and provides a practical and reliable optimized method for high-throughput gene synthesis.