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Fig. 1 | Fungal Biology and Biotechnology

Fig. 1

From: Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae

Fig. 1

Design of an artificial CRISPR gene drive system in Saccharomyces cerevisiae. a General schematic of a gene drive in a diploid genome. b An artificial gene drive system was constructed at the yeast HIS3 locus. The inducible GAL1/10 promoter drives expression of a codon-optimized S. pyogenes Cas9 containing a C-terminal eGFP fusion followed by a chosen nuclear signal sequence (NLS/NES* see Fig. 2a). An inserted terminator (from CDC10) was placed downstream of the Cas9 coding sequence followed by a selectable marker cassette. This included the non-native MX-based promoter and terminator sequences driving constitutive expression of the C. albicans URA3 gene. The entire gene drive system was flanked by two identical artificial sites, termed (u2), that do not exist in the native genome [29, 51]. The design of the drive also included an engineered “target” cassette (bottom) built within a strain of the opposite mating type at the HIS3 locus. This included an artificial “cargo” gene and a yeast-based terminator (from SHS1). A modified selectable marker cassette included the constitutive CCW12 (cell wall) promoter sequence driving the S. pombe HIS5 gene (functional equivalent to yeast HIS3). Finally, two (u1) sequences were inserted flanking the entire target locus. To complete action of the drive, a high-copy plasmid (pGF-V1220) contained the cassette for the guide RNA (marked with LEU2)

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