Fig. 2

Overview of the URA5 knockout strategies. As a proof of principle for CRISPR/Cas-mediated genetic modifications, we attempted to knockout the orotate phosphoribosyltransferase gene (URA5) to counter select 5-fluoroorotic acid (5FOA). We selected three sgRNAs from the library, which showed no off-target activities within the C. oleaginosus genome through in-silico prediction, and selectively targeted URA5. We then followed three parallel strategies to implement the Cas9 platform in C. oleaginosus. Both spheroplast batches prepared by Glucanex and HEST were used to test all strategies. a Strategy one and two: genome editing by Cas nuclease delivered into spheroplasts by electroporation in two forms separately: protein (Cas:sgRNA ribonucleoprotein [RNP]) and mRNA. In both strategies one single guide RNA (sgRNA) was used to target URA5. A single stranded DNA (ssDNA) was simultaneously transferred to introduce the repair sequences, including base deletions and base substitutions. b Strategy three: genome editing using Cas nickase as an RNP. Here, two sgRNAs targeting the leading and lagging strands were delivered to create the double-strand break (DSB). The repair ssDNA included base insertions and substitutions. A non-cutting restriction site (HindIII) was also introduced in the URA5 loci of mutants. The protospacer adjacent motifs (PAMs) were mutated in all strategies to prevent further DNA cleavage after the repair. c–e Colonies on selection agar plates with URA5 knockout using Cas mRNA, nuclease protein, and nickase protein, respectively. f The agarose gel electrophoresis of digested URA5 gene from WT and Δura5 strains. The URA5 locus was PCR amplified from the genomic DNA of mutants and WT and subjected to fast digestion by HindIII restriction enzyme. The digestion resulted in appearance of two smaller bands in the gene isolated from the Δura5 strain, indicating the integration of repair DNAs by Cas nickase. The WT URA5 gene was not digested