![]() In the second Golden Gate assembly (GG2), the sgRNA part is assembled with appropriate connectors in a second vector (AmpR-ColE1) after BsaI digestion, yielding an sgRNA cassette plasmid (white colonies growing on ampicillin plates). In the first Golden Gate assembly (GG1), partially overlapping annealed oligos containing the sgRNA sequence are cloned in the BsmBI-digested sgRNA dropout vector, yielding an sgRNA part plasmid (white colonies growing on chloramphenicol plates). The sgRNA and the Cas9 gene are cloned in a yeast expression vector through three consecutive Golden Gate assembly reactions. Schematic of the sgRNA(s) and Cas9 cloning through Golden Gate assembly In our lab, we have successfully used this procedure to generate several mutations at the SCH9 locus, encompassing full gene knockout, deletion of specific gene regions, and domain replacement ( Novarina et al., 2021), as well as to perform gene knockouts and to introduce point mutations in several yeast genes ( Guerra et al., 2021). After verification of the genome editing event(s) by PCR and/or Sanger sequencing, the Cas9+sgRNA(s) multi-gene plasmid is removed from yeast cells. Co-transformation of the Cas9+sgRNA(s) multi-gene plasmid and the repair fragment(s) in yeast results in CRISPR genome editing. One or two sgRNA are cloned in a yeast expression vector together with the Cas9 gene through three consecutive Golden Gate assembly reactions ( Engler et al., 2008 Lee et al., 2015) ( Figure 1). We provide detailed instructions for choosing the sgRNAs and designing partially overlapping complementary oligos for sgRNA cloning, as well as for the design and production of the repair fragments, depending on the nature of the desired genome editing event. This protocol describes a detailed procedure to perform CRISPR/Cas9 genome editing ( Doudna and Charpentier, 2014) in S. cerevisiae, based on the MoClo-Yeast Toolkit ( Lee et al., 2015) and a pre-existing protocol ( Akhmetov et al., 2018).
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