Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol

Oct 1, 2025Methods in molecular biology (Clifton, N.J.)

Using CRISPR to Edit Pichia pastoris Strains

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Abstract

The CRISPR/Cas9 system is a popular tool for editing genes and improving how organisms function. In this paper, we share a straightforward method for using CRISPR to make precise changes in the genome of Pichia pastoris, a type of yeast used in industry. Our protocol shows very high success in disabling specific genes, which can help scientists create better strains for various applications.

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The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.

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Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol

Abstract

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