期刊
ACS SYNTHETIC BIOLOGY
卷 4, 期 11, 页码 1226-1234出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00007
关键词
DNA assembly; CRISPR/Cas9; double-strand break; metabolic engineering
资金
- Novo Nordisk Foundation
- NNF Center for Biosustainability [Bacterial Cell Factory Optimization, Synthetic Biology Tools for Yeast, Yeast Metabolic Engineering] Funding Source: researchfish
- Novo Nordisk Fonden [NNF10CC1016517] Funding Source: researchfish
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.
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