title: Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica.
abstract: The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR-Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III-tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1'-tRNA(Gly) promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8-TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor plasmid resulted in markerless homologous recombination efficiency of over 64%. Homologous recombination was observed in 100% of transformants when nonhomologous end joining was disrupted. The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica.
authors.affiliation: Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
authors.forename: Cory M
authors.initials: CM
authors.lastname: Schwartz
authors.affiliation: Chemical and Biomolecular Engineering, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Murtaza Shabbir
authors.initials: MS
authors.lastname: Hussain
authors.affiliation: Chemical and Biomolecular Engineering, Clemson University , Clemson, South Carolina 29634, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Blenner
authors.affiliation: Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
authors.forename: Ian
authors.initials: I
authors.lastname: Wheeldon
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D059765:Homologous Recombination; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D012320:RNA Polymerase III
keywords: CRISPR−Cas9; RNA polymerase III promoters; Yarrowia lipolytica; genome editing; sgRNA expression; synthetic promoters