A versatile toolbox for CRISPR-based genome engineering in Pichia pastoris.

Pichia pastoris has gained much attention as a popular microbial cell factory for the production of recombinant proteins and high-value chemicals from laboratory to industrial scale. However, the lack of convenient and efficient genome engineering tools has impeded further applications of Pichia pastoris towards metabolic engineering and synthetic biology. Here, we report a CRISPR-based toolbox for gene editing and transcriptional regulation in P. pastoris. Based on the previous attempts in P. pastoris, we constructed a CRISPR/Cas9 system for gene editing using the RNA Pol-III-driven expression of sgRNA. The system was used to rapidly recycle the selectable marker with an eliminable episomal plasmid and achieved up to 100% knockout efficiency. Via dCas9 fused with transcriptional repressor (Mix1/RD1152) or activator (VPR), a flexible toolbox for regulation of gene expression was developed. The reporter gene eGFP driven by yeast pGAP or pCYC1 promoter showed strong inhibition (above 70%) and up to ~ 3.5-fold activation. To implement the combinatorial genetic engineering strategy, the CRISPR system contained a single Cas9-VPR protein, and engineered gRNA was introduced in P. pastoris for simultaneous gene activation, repression, and editing (CRISPR-ARE). We demonstrated that CRISPR-ARE was highly efficient for eGFP activation, mCherry repression, and ADE2 disruption, individually or in a combinatorial manner with a stable expression of multiplex sgRNAs. The simple and multifunctional toolkit demonstrated in this study will accelerate the application of P. pastoris in metabolic engineering and synthetic biology. KEY POINTS: • An eliminable CRISPR/Cas9 system yielded a highly efficient knockout of genes. • Simplified CRISPR/dCas9-based tools enabled transcriptional regulation of targeted genes. • CRISPR-ARE system achieved simultaneous gene activation, repression, and editing in P. pastoris.