TY - JOUR
T1 - High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides
AU - Cho, Namjin
AU - Seo, Han Na
AU - Ryu, Taehoon
AU - Kwon, Euijin
AU - Huh, Sunghoon
AU - Noh, Jinsung
AU - Yeom, Huiran
AU - Hwang, Byungjin
AU - Ha, Heejeong
AU - Lee, Ji Hyun
AU - Kwon, Sunghoon
AU - Bang, Duhee
N1 - Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2018/5
Y1 - 2018/5
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gky112
DO - 10.1093/nar/gky112
M3 - Article
C2 - 29529247
AN - SCOPUS:85065714222
SN - 0305-1048
VL - 46
JO - Nucleic acids research
JF - Nucleic acids research
IS - 9
M1 - e55
ER -