4.8. Mutation of Rpn4 Binding Sites Using the CRISPR/Cas9 System

Spacers were designed using CRISPOR (http://crispor.tefor.net/). Oligonucleotides encoding spacers (Table S2) were annealed and cloned into a BsaI-cut pCRCT vector [80]. Yeasts were co-transformed with pCRCT derivatives (Table S3) and PCR donor constructs. Each PCR donor carried an endonuclease (XhoI, XbaI or PstI) site instead of the GC-rich part of the PACE or PACE-like element. Yeast colonies were grown on synthetic selective media lacking uracil. Randomly picked colonies were screened for the presence of editing events using PCR followed by restriction analysis. The edited colonies were streaked to obtain single colonies, and the PACE mutations were verified by PCR followed by Sanger sequencing.