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Fig. 3 | Fungal Biology and Biotechnology

Fig. 3

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

Fig. 3

CRISPR gene drives using various NLS and NES fused to Cas9–eGFP. b Schematic of gene drive activation. Following diploid selection, yeast were grown to saturation overnight in media containing raffinose and sucrose lacking leucine. Cultures were back-diluted into rich medium containing galactose for a set number of hours, diluted to approximately 100–500 cells per agar plate (SD-LEU), and incubated for 48 h. Yeast colonies were velvet-transferred to SD-LEU and SD-HIS plates for up to 24 h before imaging. If the GD was successful and removed the target HIS3 locus (harboring SpHIS5), then colonies would be sensitive to the SD-HIS condition. b Haploid yeast strains (GFY-2756, and GFY-3465-3469) were mated to target strains (GFY-3206 and 3207), diploids selected, and gene drives activated for 5 h. Yeast were plated on SD-LEU and transferred to a final SD-LEU plate (control) and SD-HIS plate to assess gene drive activity. c The number of colonies sensitive on SD-HIS provided a measure for “percent gene drive activity”. Diploid gene drives were tested using strains from b and mutational substitutions made to each NLS (GFY-3470, 3443-3447, 3449-3452, and 3454-3456, numbered 1–17) where Cas9 was induced for either 2.5 h or 5 h and quantified for drive activity. Error, SD. NLS(I–III) sequences can be found in Fig. 2a. d Gene drive strains (GFY-3468, 3469, 3471, 3472, 2758, 3716 and 3717) harboring a NES signal in the absence or presence of additional NLSs were tested for 2.5 h, 5 h, 10 h, and 24 h of Cas9 induction and quantified as in (c). Error, SD. Red asterisk, this construct harbors the ADH1(t)-prMX-KanR-MX(t) cassette following Cas9–eGFP. e Top, illustration of the gene drive/target arrangement and the position of oligonucleotides (Additional file 1: Table S1) used. Middle, PCRs were performed on chromosomal DNA from clonal isolates from each drive (5 h). The numbers (1–17) correspond to strains from Fig. 3c. The expected sizes for each PCR ad are shown along with markers. Images were cropped from independent gels or portions of larger gels and are divided by white lines. Two isolates were obtained with no galactose activation (1’ and 2’; dextrose only treatment) from GFY-2756 (Strain 1). All colonies were tested for ploidy status (diploid) and growth on SD-URA (drive) and SD-HIS (target). Strain GFY-2756 was tested on G418 media. Below, A similar analysis of clonal isolates from the NES-containing strains was performed (24 h). Two isolates each (from strain 18 and 19) were chosen that were either resistant or sensitive to the SD-HIS condition

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