CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

Tadas Jakociunas; Arun Stephen Rajkumar; Jie Zhang; Dushica Arsovska; Edith Angelica Rodriguez Prado; Christian Bille Jendresen; Mette Louise Skjødt; Alex Toftgaard Nielsen; Irina Borodina; Michael Krogh Jensen; Jay Keasling

doi:10.1021/acssynbio.5b00007

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

Tadas Jakociunas, Arun Stephen Rajkumar, Jie Zhang, Dushica Arsovska, Edith Angelica Rodriguez Prado, Christian Bille Jendresen, Mette Louise Skjødt, Alex Toftgaard Nielsen, Irina Borodina, Michael Krogh Jensen, Jay Keasling

Novo Nordisk Foundation Center for Biosustainability

Research output: Contribution to journal › Journal article › Research › peer-review

Abstract

Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

Original language	English
Journal	A C S Synthetic Biology
Volume	4
Issue number	11
Pages (from-to)	1226-1234
Number of pages	9
ISSN	2161-5063
DOIs	https://doi.org/10.1021/acssynbio.5b00007
Publication status	Published - 2015

Bibliographical note

This paper was published to the web with an error in Figure 2e. The corrected version was reposted March 30, 2015.

Keywords

DNA assembly
CRISPR/Cas9
Double-strand break
Metabolic engineering

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Jakociunas, T., Rajkumar, A. S., Zhang, J., Arsovska, D., Rodriguez Prado, E. A., Jendresen, C. B., Skjødt, M. L., Nielsen, A. T., Borodina, I., Jensen, M. K., & Keasling, J. (2015). CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. A C S Synthetic Biology, 4(11), 1226-1234. https://doi.org/10.1021/acssynbio.5b00007

Jakociunas, Tadas ; Rajkumar, Arun Stephen ; Zhang, Jie ; Arsovska, Dushica ; Rodriguez Prado, Edith Angelica ; Jendresen, Christian Bille ; Skjødt, Mette Louise ; Nielsen, Alex Toftgaard ; Borodina, Irina ; Jensen, Michael Krogh ; Keasling, Jay. / CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. In: A C S Synthetic Biology. 2015 ; Vol. 4, No. 11. pp. 1226-1234.

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title = "CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae",

abstract = "Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.",

keywords = "DNA assembly, CRISPR/Cas9, Double-strand break, Metabolic engineering",

author = "Tadas Jakociunas and Rajkumar, {Arun Stephen} and Jie Zhang and Dushica Arsovska and {Rodriguez Prado}, {Edith Angelica} and Jendresen, {Christian Bille} and Skj{\o}dt, {Mette Louise} and Nielsen, {Alex Toftgaard} and Irina Borodina and Jensen, {Michael Krogh} and Jay Keasling",

note = "This paper was published to the web with an error in Figure 2e. The corrected version was reposted March 30, 2015.",

year = "2015",

doi = "10.1021/acssynbio.5b00007",

language = "English",

volume = "4",

pages = "1226--1234",

journal = "A C S Synthetic Biology",

issn = "2161-5063",

publisher = "American Chemical Society",

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Jakociunas, T, Rajkumar, AS, Zhang, J , Arsovska, D, Rodriguez Prado, EA, Jendresen, CB, Skjødt, ML, Nielsen, AT , Borodina, I , Jensen, MK & Keasling, J 2015, 'CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae', A C S Synthetic Biology, vol. 4, no. 11, pp. 1226-1234. https://doi.org/10.1021/acssynbio.5b00007

CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. / Jakociunas, Tadas; Rajkumar, Arun Stephen; Zhang, Jie ; Arsovska, Dushica; Rodriguez Prado, Edith Angelica; Jendresen, Christian Bille; Skjødt, Mette Louise; Nielsen, Alex Toftgaard ; Borodina, Irina ; Jensen, Michael Krogh ; Keasling, Jay.

In: A C S Synthetic Biology, Vol. 4, No. 11, 2015, p. 1226-1234.

Research output: Contribution to journal › Journal article › Research › peer-review

TY - JOUR

T1 - CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae

AU - Jakociunas, Tadas

AU - Rajkumar, Arun Stephen

AU - Zhang, Jie

AU - Arsovska, Dushica

AU - Rodriguez Prado, Edith Angelica

AU - Jendresen, Christian Bille

AU - Skjødt, Mette Louise

AU - Nielsen, Alex Toftgaard

AU - Borodina, Irina

AU - Jensen, Michael Krogh

AU - Keasling, Jay

N1 - This paper was published to the web with an error in Figure 2e. The corrected version was reposted March 30, 2015.

PY - 2015

Y1 - 2015

N2 - Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

AB - Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.

KW - DNA assembly

KW - CRISPR/Cas9

KW - Double-strand break

KW - Metabolic engineering

U2 - 10.1021/acssynbio.5b00007

DO - 10.1021/acssynbio.5b00007

M3 - Journal article

C2 - 25781611

VL - 4

SP - 1226

EP - 1234

JO - A C S Synthetic Biology

JF - A C S Synthetic Biology

SN - 2161-5063

IS - 11

ER -