CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome.

BACKGROUND: The yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality. RESULTS: We identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody. CONCLUSIONS: We demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.

Simplified Gene Knockout by CRISPR-Cas9-Induced Homologous Recombination.

Genetic engineering of industrial cell lines often requires knockout of multiple endogenous genes. Tools like CRISPR-Cas9 have enabled serial or parallelized gene disruption in a wide range of industrial organisms, but common practices for the screening and validation of genome edits are lacking. For gene disruption, DNA repair by homologous recombination offers several advantages over nonhomologous end joining, including more efficient screening for knockout clones and improved genomic stability. Here we designed and characterized a knockout fragment intended to repair Cas9-induced gene disruptions by homologous recombination. We identified knockout clones of Komagataella phaffii with high fidelity by PCR, removing the need for Sanger sequencing. Short overlap sequences for homologous recombination (30 bp) enabled the generation of gene-specific knockout fragments by PCR, removing the need for subcloning. Finally, we demonstrated that the genotype conferred by the knockout fragment is stable under common cultivation conditions.