Genome-wide CRISPR activation and repression library screening in Yarrowia lipolytica

• Jacob Nicosia, Chemical Engineering, University of Delaware

• Wilfred Chen, Chemical Engineering, University of Delaware

Yarrowia lipolytica is an oleaginous yeast that has drawn interest in industrial and research applications for its ability to produce chemicals of interest. However, its genome is not yet as thoroughly researched as that of more commonly used microbial species. To add to this knowledge base and develop a toolkit for easier bioengineering of strains of yeast with desirable phenotypes, we designed a genome-scale CRISPRa and CRISPRi library screening on Yarrowia lipolytica. The full library contains 67,426 sgRNAs; however, a pilot library containing 40 sgRNAs was worked with first to confirm small-scale success before building the full library. Cloning of pilot library constructs was done first in Escherichia coli. Two methods of assembly were compared:  direct insertion of a single-stranded oligonucleotide as well as PCR amplification of the oligonucleotide pool. Due to overamplification issues, this PCR process was optimized to determine the number of cycles that led to the greatest product yield without overamplification. After cloning in E. coli, the constructs were transformed into Yarrowia lipolytica. Here, an attempt was made to optimize transformation efficiency, as a significant number of transformants would be necessary in the full library transformation to ensure the diversity of the library. Several different transformation methods were tested, including chemical and electroporation methods, and dilution plating was used to determine how many transformants were produced. Once the pilot library was transformed into the yeast, screening experiments were performed in cultures containing canavanine or xylose. The goal of this screening was to test for possible enrichment of sgRNAs, which would indicate the gene that the sgRNA targeted was beneficial to the cell’s survival in the screening environment. Using both CRISPRa and CRISPRi allowed a self-checking approach; if repressing a gene helped the yeast survive in canavanine, activating that gene should have the opposite effect.