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Fig. 1 | Microbial Cell Factories

Fig. 1

From: SWITCH: a dynamic CRISPR tool for genome engineering and metabolic pathway control for cell factory construction in Saccharomyces cerevisiae

Fig. 1

The SWITCH strategy for cell factory construction and optimization. Step 1 The genomic engineering state is created by integrating casX. Note that a direct repeat flanks the KlURA3 marker allowing it to be recycled via direct repeat (DR) recombination. Step 2 In one transformation event several genes of interest (GOI) are simultaneously and marker-less integrated by the unified support of assembler and CasX. Step 3 The genomic engineering state is switched into the regulatory state, when CasX is directed to cleave its own gene sequence. The rescue DNA fragment contains either a codon optimized dcasX or a dcasX fused with a regulatory domain (dcasX ± RD) flanked by regions that are homologous to the integrated casX cassette. Alternatively, step 3* if the strain is finalized in step 2, the locus containing casX can be restored to wild type by the assistance of CasX and a rescue fragment containing the locus sequence. Step 4 In the regulatory state the regulator protein (dCasX or dCasX-RD) can be used to target both endogenous and heterologous GOI. Finally, after both step 3* and 4 the newly created cell factory can be characterized as part of a metabolic engineering cycle

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