The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has 12 greatly accelerated the field of strain engineering. However, insufficient efforts have been 13 made towards developing robust multiplexing tools in Saccharomyces cerevisiae. Here, we 14 exploit the RNA processing capacity of the bacterial endoribonuclease Csy4 from 15 Pseudomonas aeruginosa, to generate multiple gRNAs from a single transcript for genome 16 editing and gene interference applications in S. cerevisiae. In regards to genome editing, we 17 performed a quadruple deletion of FAA1, FAA4, POX1 and TES1 reaching 96% efficiency out 18 of 24 colonies tested. Then, we used this system to efficiently transcriptionally regulate the 3 19 genes, OLE1, HMG1 and ACS1. Thus, we demonstrate that multiplexed genome editing and 20 gene regulation can be performed in a fast and effective manner using Csy4.