Developing a CRISPR/Cas9 System for Genome Editing in the Basidiomycetous Yeast Rhodosporidium toruloides

The basidiomycetous yeast Rhodosporidium toruloides ( R. toruloides ) has been explored as a promising host for the production of lipids and carotenoids. However, the rational manipulation of this yeast remains difficult due to lack of efficient genetic tools. Here, the development of a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas9) system for genome editing in R. toruloides is described. First, R. toruloides strains are generated with sufficient production of the Cas9 protein of Staphylococcus aureus origin by integrating a cassette containing a codon‐optimized Cas9 gene into the genome. In parallel, two U6 genes are identified, predicting two U6 promoters and confirming better transcription of single‐guide RNA (sgRNA) with the U6b promoter. Next, sgRNA cassettes are designed targeting CRTI , CAR2 , and CLYBL gene, respectively, transforming into those Cas9‐expressed strains, and finding over 60% transformants with successful insertion and deletion (indel) mutations. Furthermore, when the sgRNA cassette includes donor DNA flanked by two homologous arms of the gene CRTI , gene knockout occurs via homologous recombination. Thus, the CRISPR/Cas9 system is now established as a powerful genome‐editing tool in R. toruloides , which should facilitate functional genomic study and advanced cell factory development.