High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Namjin Cho; Han Na Seo; Taehoon Ryu; Euijin Kwon; Sunghoon Huh; Jinsung Noh; Huiran Yeom; Byungjin Hwang; Heejeong Ha; Ji Hyun Lee; Sunghoon Kwon; Duhee Bang

doi:10.1093/nar/gky112

High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Namjin Cho, Han Na Seo, Taehoon Ryu, Euijin Kwon, Sunghoon Huh, Jinsung Noh, Huiran Yeom, Byungjin Hwang, Heejeong Ha, Ji Hyun Lee, Sunghoon Kwon, Duhee Bang

Department of Chemistry

Research output: Contribution to journal › Article › peer-review

4 Citations (Scopus)

Abstract

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.

Original language	English
Article number	e55
Journal	Nucleic acids research
Volume	46
Issue number	9
DOIs	https://doi.org/10.1093/nar/gky112
Publication status	Published - 2018 May

Bibliographical note

Funding Information:
Pioneer Research Center Program [NRF-2012–0009557] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Bio & Medical Technology Development Program [NRF-2016M3A9B6948494] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Center Program [NRF-2015K1A4A3047345] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Nano•Material Technology Development Program [NRF-2012M3A7A9671610] hrough the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Funding for open access charge: Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Conflict of interest statement. D.B., N.C., H.N.S. and E.K. are the authors of a patent application for the method described in this manuscript (Method for synthesizing gene using high-depth oligonucleotide tiling, US 15/184,805, 2016.06.16). The remaining authors declare they have no competing financial interest.

Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

All Science Journal Classification (ASJC) codes

Genetics

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10.1093/nar/gky112

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@article{07f9b6ec6f9148879e1908f017d88e87,

title = "High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides",

abstract = "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.",

author = "Namjin Cho and Seo, {Han Na} and Taehoon Ryu and Euijin Kwon and Sunghoon Huh and Jinsung Noh and Huiran Yeom and Byungjin Hwang and Heejeong Ha and Lee, {Ji Hyun} and Sunghoon Kwon and Duhee Bang",

note = "Funding Information: Pioneer Research Center Program [NRF-2012–0009557] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Bio & Medical Technology Development Program [NRF-2016M3A9B6948494] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Center Program [NRF-2015K1A4A3047345] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Nano•Material Technology Development Program [NRF-2012M3A7A9671610] hrough the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Funding for open access charge: Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Conflict of interest statement. D.B., N.C., H.N.S. and E.K. are the authors of a patent application for the method described in this manuscript (Method for synthesizing gene using high-depth oligonucleotide tiling, US 15/184,805, 2016.06.16). The remaining authors declare they have no competing financial interest. Publisher Copyright: {\textcopyright} The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.",

year = "2018",

month = may,

doi = "10.1093/nar/gky112",

language = "English",

volume = "46",

journal = "Nucleic Acids Research",

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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 - Funding Information: Pioneer Research Center Program [NRF-2012–0009557] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Bio & Medical Technology Development Program [NRF-2016M3A9B6948494] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Center Program [NRF-2015K1A4A3047345] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning; Nano•Material Technology Development Program [NRF-2012M3A7A9671610] hrough the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Funding for open access charge: Mid-career Researcher Program [NRF-2015R1A2A1A10055972] through the National Research Foundation of Korea, funded by the Ministry of Science, ICT & Future Planning. Conflict of interest statement. D.B., N.C., H.N.S. and E.K. are the authors of a patent application for the method described in this manuscript (Method for synthesizing gene using high-depth oligonucleotide tiling, US 15/184,805, 2016.06.16). The remaining authors declare they have no competing financial interest. 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.

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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 -

High-throughput construction of multiple cas9 gene variants via assembly of high-depth tiled and sequence-verified oligonucleotides

Abstract

Bibliographical note

All Science Journal Classification (ASJC) codes

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