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CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae
- Source :
- ACS synthetic biology. 4(11)
- Publication Year :
- 2015
-
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.
- Subjects :
- Genetics
Cas9
Saccharomyces cerevisiae
Biomedical Engineering
General Medicine
Biology
biology.organism_classification
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Genome engineering
chemistry.chemical_compound
Plasmid
chemistry
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
Synthetic Biology
Genome, Fungal
Homologous recombination
DNA, Fungal
Gene
DNA
Subjects
Details
- ISSN :
- 21615063
- Volume :
- 4
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- ACS synthetic biology
- Accession number :
- edsair.doi.dedup.....c7434da82eb482f302f393bbc0a7b5f4