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
SN - 2161-5063
VL - 4
SP - 1226
EP - 1234
JO - A C S Synthetic Biology
JF - A C S Synthetic Biology
IS - 11
ER -