The oleaginous yeast Yarrowia lipolytica has been a focus of metabolic engineering due to its ability to produce and modify alkanes and lipids. Despite a number of successes, genetic manipulations including gene disruptions and markerless integrations are challenging. Current studies have relied on targeted integration of markered cassettes to disrupt gene function and integrate expression cassettes, resulting in depletion of available selectable markers. A Cre-Lox recombinase system has been developed and used for marker recovery, but it is difficult to adapt for rapid strain generation. To facilitate rapid genome engineering, we adapted the CRISPR/Cas9 system from Streptococcus pyogenes for use in Y. lipolytica. To achieve high editing efficiencies, several native and synthetic RNA polymerase III promoters were investigated for sgRNA expression. A synthetic promoter consisting of the SCR1 promoter concatenated with a tRNA achieved disruption of a target gene via indel formation at a rate of 95%. We used this synthetic promoter to express sgRNA and combined it with a Y. lipolytica codon optimized Cas9 to yield the pCRISPRyl system, a simple to clone single plasmid for double strand break generation in Y. lipolytica. To facilitate the use of this pCRISPRyl system to induce homologous recombination (HR), three genes involved in nonhomologous end joining (NHEJ) were disrupted. We found that disruption of KU70, KU80, and DNL4 all had similar effects on the rate of HR of a transformed linear fragment. When the pCRISPRyl system was used to generate a double strand break in the NHEJ deficient strains without a HR repair template, sequencing revealed that microhomology mediated end joining was likely responsible for double strand break repair. The pCRISPRyl system was then used in conjunction with a HR repair template to allow for targeted markerless gene insertion in Y. lipolytica.

High efficiency genome editing in Yarrowia lipolytica by CRISPR/Cas9

251st National Meeting (2016)

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