Chapter title |
Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR
|
---|---|
Chapter number | 12 |
Book title |
Synthetic Metabolic Pathways
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7295-1_12 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7294-4, 978-1-4939-7295-1
|
Authors |
Tadas Jakočiūnas, Emil D. Jensen, Michael K. Jensen, Jay D. Keasling |
Abstract |
Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building. |
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Mendeley readers
Geographical breakdown
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Researcher | 6 | 21% |
Student > Doctoral Student | 3 | 10% |
Professor > Associate Professor | 3 | 10% |
Student > Master | 2 | 7% |
Other | 4 | 14% |
Unknown | 4 | 14% |
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Engineering | 2 | 7% |
Immunology and Microbiology | 1 | 3% |
Chemical Engineering | 1 | 3% |
Other | 0 | 0% |
Unknown | 4 | 14% |