Genome Editing in Yarrowia Lipolytica Using a Crispr-Cas9 System with RNA Polymerase II Transcribed Single Guide RNA

Yarrowia lipolytica has been the focus of a wide range of studies due to its ability to make and accumulate lipids to a high level.  Here, we engineer a single plasmid CRISPR-Cas9 system for Y. lipolytica that enables markerless gene knockouts and insertions.  A functional system was created using a codon optimized Cas9 from Streptococcus pyogenes and single guide RNA (sgRNA) expressed from the well characterized constitutive TEF promoter.  RNA cleavage by flanking ribozymes generated the mature sgRNA, ensuring proper folding and preventing export to the cytosol by mRNA transport processes.  Gene knockout efficiency of ≥75% was achieved when targeting the ku70 gene without a cotransformed homologous recombination (HR) donor.  The ku70 gene was targeted as it has been shown that Y. lipolytica strongly favors non-homologous end-joining (NHEJ) to repair double strand breaks.  Knockout of ku70 suppresses NHEJ and allows HR to serve as the primary double strand break repair mechanism.  Using the generated Δku70 strain and the CRISPR-Cas9 system, markerless gene tagging with fluorescent proteins is being done to investigate protein localization to lipid droplets and to the endoplasmic reticulum.