A fast and reliable Golden Gate-based cloning strategy, which allows us to target the Cas9 protein (molecular scissors) to multiple different locations throughout the genome, where it cuts double-stranded DNA to enable targeted insertions or deletions of DNA sequences. By using a nuclease deficient dCas9 protein, this system can also be used to up- or downregulate transcriptional levels of multiple genes in parallel.
Synthetic Sc2.0 chromosomes harbor symmetrical recombination sites (loxPsym) downstream of every non-essential gene. This enables to SCRaMbLE the whole genetic background in order to generate an optimized host organism for specific pathway engineering projects.
In our group, we established a red-light recombination system, dubbed L-SCRaMbLE, perfectly suited for the recombination and optimization of the yeast genome.