Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris

Astrid Weninger, Anna-Maria Hatzl, Christian Schmid, Thomas Vogl, Anton Glieder

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is one of the most commonly used expression systems for heterologous protein production. However the recombination machinery in P. pastoris is less effective in contrast to Saccharomyces cerevisiae, where efficient homologous recombination naturally facilitates genetic modifications. The lack of simple and efficient methods for gene disruption and specifically integrating cassettes has remained a bottleneck for strain engineering in P. pastoris. Therefore tools and methods for targeted genome modifications are of great interest. Here we report the establishment of CRISPR/Cas9 technologies for P. pastoris and demonstrate targeting efficiencies approaching 100%. However there appeared to be a narrow window of optimal conditions required for efficient CRISPR/Cas9 function for this host. We systematically tested combinations of various codon optimized DNA sequences of CAS9, different gRNA sequences, RNA Polymerase III and RNA Polymerase II promoters in combination with ribozymes for the expression of the gRNAs and RNA Polymerase II promoters for the expression of CAS9. Only 6 out of 95 constructs were functional for efficient genome editing. We used this optimized CRISPR/Cas9 system for gene disruption studies, to introduce multiplexed gene deletions and to test the targeted integration of homologous DNA cassettes. This system allows rapid, marker-less genome engineering in P. pastoris enabling unprecedented strain and metabolic engineering applications.

Original languageEnglish
Pages (from-to)139-49
Number of pages11
JournalJournal of Biotechnology
Volume235
DOIs
Publication statusPublished - 10 Oct 2016

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Clustered Regularly Interspaced Short Palindromic Repeats
Pichia
Guide RNA
RNA Polymerase II
Yeasts
Genome
RNA Polymerase III
Metabolic Engineering
Catalytic RNA
Homologous Recombination
Gene Deletion
Codon
Genetic Recombination
Genes
Saccharomyces cerevisiae
Technology
DNA
Proteins

Keywords

  • Journal Article
  • Review

Cite this

Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. / Weninger, Astrid; Hatzl, Anna-Maria; Schmid, Christian; Vogl, Thomas; Glieder, Anton.

In: Journal of Biotechnology, Vol. 235, 10.10.2016, p. 139-49.

Research output: Contribution to journalArticleResearchpeer-review

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