“…Another major limitation is the relatively low copy numbers of gRNA expressing plasmid in industrial yeast strains. So far, several strategies have been adopted for the expression of Cas9 and gRNA and most of the studies have found it necessary to express gRNA on a 2 μ based multicopy plasmid for efficient genome engineering in S. cerevisiae (DiCarlo et al, ; Stovicek et al, ; Stovicek, Holkenbrink, & Borodina, ). Interestingly, although the 2 μ‐based plasmids generally have relatively high copy numbers (20–30 copies/cell) with auxotrophic markers (i.e., HIS3 , TRP1 , LEU2 , and URA3 ), they can be as low as 3–5 copies/cell when antibiotic markers (i.e., KanMX and HygB ) are included (Karim, Curran, & Alper, ; Lian, Jin, & Zhao, ).…”