Abstract

The clustered regularly interspaced short palindromic repeats-associated protein 9 technology stands out as the paramount gene-editing tool owing to its unparalleled efficiency and precision. It has rapidly been applied to various biological systems, including bacteria, plants and yeasts. P. pastoris (now known as Komagataella phaffii) is a commonly used industrial microorganism with advantages such as rapid growth, high-density culture, robust expression of exogenous proteins, with its simplicity in genomic manipulation, clustered regularly interspaced short palindromic repeats-associated protein 9 renders itself as an optimal platform for synthesizing recombinant proteins, enzymes and various biologics. The application of clustered regularly interspaced short palindromic repeats-associated protein 9 in P. pastoris focuses on gene editing and bio manufacturing, enabling precise modifications of its genome to enhance metabolic pathways and improve product quality. Recent advancements have demonstrated significant progress utilizing clustered regularly interspaced short palindromic repeats-associated protein 9 for metabolic engineering and protein manipulation in P. pastoris, substantially increasing the yield of target metabolites and recombinant proteins. Despite these advances, technical challenges such as off-target effects and unstable editing efficiency remain. Therefore, further optimization of the clustered regularly interspaced short palindromic repeatsassociated protein 9 system, development of new Cas variants, and improvement in guide ribonucleic acid design are current research hotspots and challenges. This review systematically summarizes the progress of clustered regularly interspaced short palindromic repeats-associated protein 9 application in P. pastoris, focusing on specific application examples and technology optimization strategies in gene editing and bio manufacturing. It also discusses the current challenges and future research directions. The aim is to provide researchers with a comprehensive and in-depth knowledge framework to promote the further development and application of this technology in bio manufacturing, stimulate more innovative research ideas, and bring new breakthroughs in the genetic engineering and bio manufacturing of P. pastoris.