Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Abstract

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ∼20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.

Pichia pastoris protease‐deficient and auxotrophic strains generated by a novel, user‐friendly vector toolbox for gene deletion

“…We envision our deletion plasmids to be used to generate knockout strains that can be mated and analysed by tetrad dissection. We further suggest to use our vectors in combination with a recently developed CRISPR/Cas9 approach (Weninger et al, ), which still requires the cloning of repair cassettes of several kbs. Genome editing of P. pastoris applying CRISPR/Cas9 is working much more efficiently in a ku70 Δ strain background than in wild type cells, which is why the use of efficiently working selection markers in the repair cassettes drastically facilitates identification of strains with correct genetic alterations.…”

“…Several strategies were shown to improve HR activity in P. pastoris such as hydroxyurea‐mediated cell cycle arrest (Tsakraklides et al, ), increasing the genetic redundancy of host cells by providing extra copies of the gene to be deleted on a helper plasmid (Chen et al, ) or deletion of KU70 , a key player in NHEJ improved targeting (Näätsaari et al, ). Deletion of KU70 was also shown to drastically improved gene modifications using CRISPR/Cas9 tools (Weninger et al, ). However, disadvantages such as high complexity, low strain fitness, or not yet fully realized methods for donor cassette integration have kept these techniques from replacing the established ones for the efficient construction of producer strains.…”

Pichia pastoris as a Versatile Cell Factory for the Production of Industrial Enzymes and Chemicals: Current Status and Future Perspectives

“…Stadlmayr et al reported the screening of highly secreting strains by constructing cDNA overexpression libraries but only one gene per strain was perturbed in this strategy so that the coordination between genes could not be examined. Therefore, more efficient genome‐scale perturbation methods such as gTME (global transcription machinery engineering) , MAGE (multiplex automated genome engineering), GREASE (genome replication engineering‐assisted continuous evolution), and more recently CRISPR/Cas9 will pave the way for the modification of complex phenotypes such as secretion capability and cell robustness.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…The discovery of the CRISPR‐Cas9 genome editing system has revolutionized synthetic biology by allowing fast genetic manipulation in a variety of applications. CRISPR‐Cas9 has been implemented in a variety of S. cerevisiae backgrounds, including strains used in the brewing and wine industries (Denby et al, ; Gorter de Vries, de Groot, van den Broek, & Daran, ; Vigentini, Gebbia, Belotti, Foschino, & Roth, ) and other industrially relevant yeast species such as fission yeasts (Jacobs, Ciccaglione, Tournier, & Zaratiegui, ), Kluyveromyces marxianus (Lee et al, ), Yarrowia lipolityca (Schwartz & Wheeldon, ; Shi, Huang, Kerkhoven, & Ji, ), Ogatae species (Juergens et al, ), and Pichia species (Weninger et al, ). Of particular relevance is the development of optimized CRISPR‐Cas9 methods to engineer diploid and polyploid industrial yeast strains (Stovicek, Borja, Forster, & Borodina, ; Zhang et al, ).…”