Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis.  Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system.  The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets.  The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.
CRISPR/Cas9
mSphere
D064113:CRISPR-Cas Systems; D017353:Gene Deletion; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae; D004718:Saccharomycetales
Tran Vinh G VG ;Cao Mingfeng M ;Fatma Zia Z ;Song Xiaofei X ;Zhao Huimin H 