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

Journal of Biotechnology
Astrid WeningerAnton Glieder

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 CRIS...Continue Reading

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Related Concepts

Biological Markers
Establishment and Maintenance of Localization
RNA Polymerase II
RNA Polymerase III
Non-Homologous DNA End-Joining
Untranslated Regions
Glycerin
Genome
DNA Repair
Genes

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