Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica

ACS Synth Biol. 2016 Apr 15;5(4):356-9. doi: 10.1021/acssynbio.5b00162. Epub 2016 Jan 7.


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.

Keywords: CRISPR−Cas9; RNA polymerase III promoters; Yarrowia lipolytica; genome editing; sgRNA expression; synthetic promoters.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Fungal Proteins / antagonists & inhibitors
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Gene Editing / methods*
  • Homologous Recombination
  • Plasmids / genetics
  • Plasmids / metabolism
  • Promoter Regions, Genetic
  • RNA Polymerase III / genetics*
  • RNA, Guide / metabolism
  • Real-Time Polymerase Chain Reaction
  • Yarrowia / genetics
  • Yarrowia / metabolism*


  • Fungal Proteins
  • RNA, Guide
  • RNA Polymerase III