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GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris. Prielhofer R, Barrero JJ, Steuer S, Gassler T, Zahrl R, Baumann K, Sauer M, Mattanovich D, Gasser B, Marx H. BMC Syst Biol. (2017) 11,123. doi: 10.1186/s12918-017-0492-3. PubMed PMID: 29221460.
The CRISPi Kit contains basic elements for performing CRISPR/Cas9 mediated gene and genome editing in the yeast P. pastoris. The kit contains seven different backbone 3 (BB3) vectors with functional Cas9 and sgRNA expression cassettes and three different selection markers.
Each CRISPi BB3 carries:
S. cerevisiae CEN6/ARS4 sequence for episomal plasmid maintenance, enabling the recovery of plasmid free cells after successful genome engineering
Human codon-optimized hCas9 gene fused to a nuclear localization signal (NLS) under control of a constitutive promoter (original Cas9 sequence - Addgene #43802)
An insertion site for the sgRNA with flanking ribozyme sequences under control of the P. pastoris PGAP promoter
A marker cassette for yeast/E. coli selection natMX (nourseothricin (NTC) selection), hphMX (hygromycin B) or kanMX (geneticin (G418))
All plasmids were assembled using the GoldenPiCS Kit (Addgene Kit #1000000133).
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The CRISPi Kit was a gift from Brigitte Gasser (Addgene Kit #1000000136).”