CRISPR-Cas9-based one-step multiplexed genome editing through optimizing guide RNA processing strategies in Pichia pastoris.

ABSTRACT: The important methylotrophic yeast Pichia pastoris has been utilized for the production of a variety of heterologous recombinant proteins and has great potential for use in the production of value-added compounds using methanol as a substrate. However, the lack of convenient and efficient genome engineering tools has hindered further applications of P. pastoris, especially in complex and multistep metabolic engineering scenarios. Hence, we developed a rapid and convenient multi-gene editing system based on CRISPR/Cas9 by optimizing the guide RNA processing strategy, which can achieve dual-gene knockout or multi-gene integration in single step. Firstly, we found that the HgH (HH-sgRNA-HDV) structure achieved the highest single-gene knockout efficiency (95.8 %) among the three

SUBMITTER: Chen K 

PROVIDER: S-EPMC11847659 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature