(194ac) CRISPR–Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis

Scheffersomyces stipitis is an important nonconventional yeast known for its strong native xylose-utilizing capacity and has been recently shown as a well-suited host to produce compounds derived from shikimate pathway. However, its significant industrial application is hampered by the lack of genetic engineering tools. Here we developed CRISPR-Cas9 system in S. stipitis by using a codon-optimized Cas9 from Streptococcus pyogenes and single guide RNA (SgRNA), which improved targeted indel knockout efficiency by at least 80-fold. We further modified the CRISPR system via shortening SNR52 promoter for easy-cloning and improving electroporation voltage for high transformation efficiency. Then, we constructed ku genes deficient strain and succeeded in increasing homologous recombination (HR)-based genome editing efficiency by at least 2-fold. Furthermore, we developed a transcription-modulating tool using CRISPR-dCas9 and validated that the repressor Mxi1 could promote the function of dCas9 to repress gene expression by 33%. Finally, we implemented our CRISPR system in the shikimate pathway, tuned gene expression, and improved shikimate and its derivatives production. Our methods enable delicately designed genetic/metabolic manipulations in S. stipitis, and provided good references for developing efficient CRISPR-Cas9/dCas9 tools in other nonconventional yeasts.