Komagataella phaffii (Pichia pastoris) is an attractive alternative host for biopharmaceutical manufacturing, boasting high growth rates and high protein secretory capacity. The small genome (~10 Mbp) enables cheap, high throughput –omics-based characterization for genome-wide understanding of host biology and product expression. With such easily accessible biological understanding, this and other microbial hosts are amenable to extensive genome engineering. To date, the most extensive feat of genome engineering in K. phaffii is the humanization of product glycosylation by Glycofi Inc. (now Merck), which required many full time efforts over many years. The resulting engineered strain exhibited slow growth due to ~10 heterologous genes and several recycled auxotrophic markers. Today, tools like CRISPR-Cas9 obviate the need for markers and enable development of such a strain in just a few months. CRISPR-Cas9 has quickly gained popularity in model organisms due to its near universal effectiveness and modularity. Multiplexed genome edits via CRISPR-Cas9 require a scheme for modular expression of guide RNA. Current state of the part in K. phaffii relies on self-cleaving ribozyme sequences to trim mature mRNA into guide RNA. While reliable, this strategy greatly increases the variable region for gene targeting, reducing modularity and increasing the cost of high throughput CRISPR screens. Here, we demonstrate the use of –omics to inform an improved platform for CRISPR in microbial hosts. We used small-RNA sequencing to select and test RNA polymerase III promoters for modular guide RNA expression, and traditional RNA sequencing to rationally tune the expression of Cas9. We demonstrate rapid, modular genome editing of several genes on the order of weeks. This –omics based approach represents a generalizable strategy to create a reliable platform for CRISPR-Cas9 in non-model organisms.

Transcriptomics informs simplified CRISPR/Cas9 genome editing in Pichia pastoris

2019 Spring National Meeting

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