The Saccharomyces cerevisiae strain used in the CAN1 mutagenesis analysis of the CRISPR system and the gRNA plasmid/donor DNA transformation in Cas9-expressing cells was BY4733 (MATa his3Δ200 trp1Δ63 leu2Δ0 met15Δ0 ura3Δ0), which was a kind gift from Fred Winston. Parental BY4733 was grown in YPAD before transformation and then propagated in the appropriate synthetic complete (SC) media minus the auxotrophic compound complemented by the plasmids. Strain VL6-48 (MATα, his3Δ200, trplΔ1, ura3-52, ade2-101, lys2, psio, cir°) was used for the homologous recombination experiments using the gRNA PCR product, owing to its native ade2-101 premature stop codon.VL6-48 was purchased from ATCC (MYA-3666). Plasmids p415-Gal-L and p426-Gal1 used in this study were a kind gift from Fred Winston (15 (link)).
Protocol detail
CRISPR-mediated Mutagenesis in Yeast Strains
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Crispr
Donor
Homologous recombination
Mutagenesis
Parental
Plasmids
Premature stop codon
Saccharomyces cerevisiae
Strain
Corresponding Organization :
Other organizations : Boston University, Harvard University
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Variable analysis
independent variables
- CRISPR system
- GRNA plasmid/donor DNA transformation in Cas9-expressing cells
- CAN1 mutagenesis analysis
- Saccharomyces cerevisiae strain BY4733 (MATa his3Δ200 trp1Δ63 leu2Δ0 met15Δ0 ura3Δ0)
- Strain VL6-48 (MATα, his3Δ200, trplΔ1, ura3-52, ade2-101, lys2, psi⁰, cir°)
- Plasmids p415-Gal-L and p426-Gal1
- Parental BY4733 strain grown in YPAD before transformation
- Not explicitly mentioned
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