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

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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 languageEnglish
JournalA C S Synthetic Biology
Volume4
Issue number11
Pages (from-to)1226-1234
Number of pages9
ISSN2161-5063
DOIs
Publication statusPublished - 2015

Bibliographical note

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

CitationsWeb of Science® Times Cited: No match on DOI

    Research areas

  • DNA assembly, CRISPR/Cas9, Double-strand break, Metabolic engineering

ID: 108343695