Saccharomyces cerevisiae IMX2713 (CEN.PK113-7D deltasga1::cas9-natNT2 deltaX-2::Ercas12a)

ErCas12a is a class II type V CRISPR-Cas nuclease isolated from Eubacterium rectale with attractive fundamental characteristics, such as RNA self-processing capability, and also lacks reach-through royalties typical for Cas nucleases. This study aims to develop a ErCas12a-mediated genome editing tool applicable in the model yeast Saccharomyces cerevisiae. The optimal design parameters for ErCas12a editing in S. cerevisiae are defined as a 21 nt spacer flanked by 19 nt direct repeats expressed from either RNApolII or III promoters, ultimately achieving near 100% editing efficiencies in commonly targeted genomic locations. To be able to transfer the ErCas12a genome editing tool to different strain lineages, a transportable platform plasmid was constructed and evaluated for its genome editing efficiency. Using an identical crRNA expression design, the transportable ErCas12a genome editing tool showed lower efficiency when targeting the ADE2 gene. In contrast to genomic Ercas12a expression, episomal expression of Ercas12a decreases maximum specific growth rate on glucose, indicating ErCas12a toxicity at high expression levels. Moreover, ErCas12a processed a multi-spacer crRNA array using the RNA self-processing capability and this feature allowed for simultaneous editing of multiple chromosomal locations. ErCas12a is a valuable addition to the genetic toolbox for S. cerevisiae.The goal of the present study was to optimize the ErCas12a-based genome editing in S. cerevisiae. To this end, we explored ways to improve the efficiency of genome editing by tuning the length of the direct repeats and of the spacers. We also evaluated approaches to express the crRNA array by testing different designs and consequently proposed design principles for application of ErCas12a in S. cerevisiae. Furthermore, we applied these principles to investigate applicability of ErCas12a to multiplexing strategies.