(254g) CRISPR-Enabled Functional Genomics in the Oleaginous Yeast Yarrowia Lipolytica (Invited Speaker)

Due to its ability to synthesize and accumulate intracellular lipids to high levels, Yarrowia lipolytica has become one of the most studied microorganisms for the biosynthesis of oleochemicals. In this presentation, we describe our efforts in designing and creating functional genomics tools to study and engineer this yeast. The key concept that we describe is the ability to characterize the double strand break cutting efficiency of CRISPR-Cas9 across the genome of Y. lipolytica. The cutting score (CS) metric that results from these experiments enables high quality genome-wide screening hits for desired phenotypes as well as the identification essential genes under given growth conditions. Defining a CS for over 48,000 single guide RNAs (sgRNAs) also enabled the high confidence prediction of effective sgRNAs through machine learning. Combined, our experiments and predictive capabilities provide a thorough description of the functioning of CRISPR-Cas9 across the Y. lipolytica genome, creates a list of effective sgRNAs for any gene in the genome, and enables the ability to predict new sgRNAs with high confidence.