Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

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

PROVIDER: PRJNA1061507 | ENA |

REPOSITORIES: ENA

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	SRR27441410_1.fastq.gz	Fastqsanger.gz
	SRR27441410_2.fastq.gz	Fastqsanger.gz
	SRR27441411_1.fastq.gz	Fastqsanger.gz
	SRR27441411_2.fastq.gz	Fastqsanger.gz
	SRR27441412_1.fastq.gz	Fastqsanger.gz

Items per page:

1 - 5 of 24

Similar Datasets

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

Project description:The yeast Komagataella phaffii is a promising alternative host for manufacturing of therapeutic proteins. Deletion of unneeded endogenous proteins could increase the secreted titer of recombinant proteins by redirecting cellular resources. Genetic engineering in non-model hosts is hampered by limited annotation of genes, especially essential genes. In this study, we identified the set of endogenous secreted proteins in K. phaffii and attempted to disrupt these genes. We designed, transformed, and sequenced a pooled CRISPR-Cas9 knockout library to determine which genes are essential. With this knowledge, we rapidly disrupted up to 9 consecutive genes in K. phaffii. Engineered strains exhibited a ~20x increase in the production of human serum albumin and a 2x increase in the production of a monoclonal antibody. The pooled CRISPR-Cas9 library and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant therapeutic proteins and enzymes.

2024-08-08 | GSE252605 | GEO

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

Project description: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. 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. 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.

| S-EPMC11293167 | biostudies-literature

Komagataella phaffii

Project description:Komagataella phaffii SNP analysis

| PRJNA592463 | ENA

Komagataella phaffii

Project description:Komagataella phaffii Raw sequence reads

| PRJNA1157989 | ENA

Komagataella phaffii

Project description:Komagataella phaffii Raw sequence reads

| PRJNA930494 | ENA

Komagataella phaffii

Project description:Komagataella phaffii Transcriptome or Gene expression