Biology subjects

Pedersen, L. E.

Publications and source records attributed to Pedersen, L. E..

9 recordsLinked to original sources

bioRxiv2026

Glycoengineering of extracellular vesicles enhances cellular uptake and cargo delivery to target cells

The glycocalyx is a major regulator of membrane recognition, yet its specific influence on extracellular vesicles (EVs) cellular uptake remains poorly defined. We established a genetic glycoengineering platform to systematically investigate how the major glycan classes on small EVs (sEVs) modulate cell interactions and functional cargo delivery. Using an isogenic panel of HEK293F lines lacking distinct glycan biosynthetic pathways, we find that removing glycosaminoglycans ({Delta}GAG-sEVs) yields a strong increase in cellular uptake and delivery of diverse cargos, including DNA oligonucleotides, siRNA, proteins, and plasmid DNA. Glycan-modified recipient cells show that sEV-cell communication and internalization is jointly governed by glycan features on both membranes. {Delta}GAG-sEVs strongly improve gene delivery and expression in recipient cells and in a physiologically relevant human airway epithelial model. These findings establish glycan structures as tunable regulators of sEV uptake and position {Delta}GAG-sEVs as potent vehicles for improved drug delivery and gene therapy.

bioengineering↗

bioRxiv2022

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

bioengineering↗

bioRxiv2021

A synthetic RNA-mediated evolution system in yeast

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in bakers yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.

bioengineering↗

bioRxiv2020

Synergistic stabilization of a double mutant in CI2 from an in-cell library screen

Most single point mutations destabilize folded proteins. Mutations that stabilize a protein typically only have a small effect and multiple mutations are often needed to substantially increase the stability. Multiple point mutations may act synergistically on the stability, and it is not straightforward to predict their combined effect from the individual contributions. Here, we have applied an efficient in-cell assay to select variants of the barley chymotrypsin inhibitor 2 with increased stability. We find two variants that are more than 3.8 kJ/mol more stable than the wild-type. In one case the increased stability is the effect of the single substitution D55G. The other case is a double mutant, L49I/I57V, which is 5.1 kJ/mol more stable than the sum of the effects of the individual mutations. In addition to demonstrating the strength of our selection system for finding stabilizing mutations, our work also demonstrate how subtle conformational effects may modulate stability.

biophysics↗

bioRxiv2020

A dual-reporter system for investigating and optimizing protein translation and folding in E. coli

Strategies for investigating and optimising the expression and folding of proteins for biotechnological and pharmaceutical purposes are in high demand. Here, we describe a dual-reporter biosensor system that simultaneously assesses in vivo protein translation and protein folding, thereby enabling rapid screening of mutant libraries. We have validated the dual-reporter system on five different proteins and find an excellent correlation between reporter intensity signals and the levels of protein expression and solubility of the proteins. We further demonstrate the applicability of the dual-reporter system as a screening assay for deep mutational scanning experiments. The system enables high throughput selection of protein variants with high expression levels and altered protein stability. Next generation sequencing analysis of the resulting libraries of protein variants show a good correlation between computationally predicted and experimentally determined protein stabilities. We furthermore show that the mutational experimental data obtained using this system may be useful for protein structure calculations.

biochemistry↗

bioRxiv2020

Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

Pooled CRISPR screens have been widely applied in mammalian cells to discover genes regulating various phenotypes of interest. In such screens, CRISPR components are generally delivered with a lentivirus. However, lentiviral CRISPR screens are limited by unpredictable genome insertion, the requirement of biosafety level II lab facilities and personnel trained to work with viruses. Here we established a virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 74,617 gRNAs targeting 18,353 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad thus reducing the clonal variation. Using this VF CRISPR screening platform, 338 genes are identified as essential for CHO cell growth and 76 genes were found to be involved in the unfolded protein response (UPR) induced endoplasmic reticulum (ER) stress. Extensive validation of the candidate genes further demonstrated the robustness of this novel non-viral CRISPR screen method.

genomics↗

bioRxiv2020

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Over the past decades, optimization of media formulation and feeding strategies have fueled a many-fold improvement in CHO-based biopharmaceutical production. While Design of Experiments (DOE) and media screens have led to many advances, genome editing offers another avenue for enhancing cell metabolism and bioproduction. However the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we developed a comprehensive pooled CRISPR screen for CHO cell metabolism, including [~]16,000 gRNAs against [~]2500 metabolic enzymes and regulators. We demonstrated the value of this screen by identifying a glutamine response network in CHO cells. Glutamine is particularly important since it is often substantially over-fed to drive increased TCA cycle flux but can lead to accumulation of toxic ammonia. Within the glutamine-response network, the deletion of a novel and poorly characterized lipase, Abhd11, was found to substantially increase growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait.

bioengineering↗

bioRxiv2019

Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a \"clean\" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

bioengineering↗

bioRxiv2019

Adaptive laboratory evolution reveals general and specific chemical tolerance mechanisms and enhances biochemical production

Tolerance to high product concentrations is a major barrier to achieving economically viable processes for bio-based chemical production. Chemical tolerance mechanisms are often unknown, thus their rational design is not achievable. To reveal unknown tolerance mechanisms we used an automated platform to evolve Escherichia coli to grow in previously toxic concentrations of 11 chemicals that have applications as polymer precursors, chemical intermediates, or biofuels. Re-sequencing of isolates from 88 independently evolved populations, reconstruction of mutations, and cross-compound tolerance profiling was employed to uncover general and specific tolerance mechanisms. We found that: 1) the broad tolerance of strains towards chemicals varied significantly depending on the chemical stress condition under which the strain was evolved; 2) the strains that acquired high levels of NaCl tolerance also became broadly tolerant to most chemicals; 3) genetic tolerance mechanisms included alterations in regulatory, cell wall, transcriptional and translational functions, as well as more chemical-specific mechanisms related to transport and metabolism; 4) using pre-tolerized starting strains can significantly enhance subsequent production of chemicals when a production pathway is inserted; and 5) only a subset of the evolved isolates showed improved production indicating that this approach is especially useful when a large number of independently evolved isolates are screened for production. We provide a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 224 chemical tolerant strains.

microbiology↗