High efficiency CRISPR/Cas9 genome editing system with an eliminable episomal sgRNA plasmid in Pichia pastoris.

Pichia pastoris is a methylotrophic yeast in which host heterologous expression of proteins has been developed owing to the strong inducible alcohol oxidase promoter (PAOX1). However, it is difficult to manipulate the genome in P. pastoris. Based on previous attempts to apply the CRISPR/Cas9 system in P. pastoris, a CRISPR/Cas9 system with episomal sgRNA plasmid was developed and 100 % genome editing efficiency, high multicopy gene editing and stable multigene editing were obtained without a sharp decline caused by multi-sgRNA. And 28/34 (∼82 %) sgRNAs tested were effective. The CGG may have a slightly higher and more stable cleavage efficiency than the other three NGG motifs, and a low GC content may be preferable for higher cleavage efficiency. This provides researchers with a stable genome editing tool that shows a high editing efficiency, shortening the experimentation period. Furthermore, we introduced dCas9 into P. pastoris and achieved target gene interference, expanding the CRISPR/Cas9 toolbox in P. pastoris.