An innovative pooled CRISPR/AsCpf1 screen using paired gRNAs in Chinese Hamster Ovary cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB35876 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR3771220_1.fastq.gz | Fastqsanger.gz | |||
| ERR3771220_2.fastq.gz | Fastqsanger.gz | |||
| ERR3771490.fastq.gz | Fastqsanger.gz | |||
| ERR3771221_1.fastq.gz | Fastqsanger.gz | |||
| ERR3771221_2.fastq.gz | Fastqsanger.gz |
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Project description:Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins. Recently, significant progress has been made due to advances in genome sequence and annotation quality to unravel the black box CHO. Nevertheless, in many cases the link between genotype and phenotype in the context of suspension cultivated production cell lines is still not fully understood. While frameshift approaches targeting coding genes are frequently used, the non-coding regions of the genome have received less attention with respect to such functional annotation. Importantly, for non-coding regions frameshift knock-out strategies are not feasible. In this study, we developed a CRISPR-mediated screening approach that performs full deletions of genomic regions to enable the functional study of both the translated and untranslated genome. An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was established and used to generate a library tackling process-related genes and long non-coding RNAs. Next generation sequencing analysis of the plasmid library revealed a sufficient, but highly variable pgRNA composition. Recombinase-mediated cassette exchange was applied for pgRNA library integration rather than viral transduction to ensure single copy representation of pgRNAs per cell. After transient AsCpf1 expression, cells were cultivated over two sequential batches to identify pgRNAs which massively affected growth and survival. By comparing pgRNA abundance, depleted candidates were identified and individually validated to verify their effect.
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Project description:The dataset set consists of 295 microarrays from 121 individual CHO cultures producing a range of biologics including monoclonal antibodies, fusion proteins and therapeutic factors; non-producing cell lines were also included. Samples were taken from a wide range of process scales and formats that varied in terms of seeding density, temperature, medium, feed medium, culture duration and product type. Cells were sampled for gene expression analysis at various stages of the culture and bioprocess-relevant characteristics including cell density, growth rate, viability, lactate, ammonium and cell specific productivity (Qp) were determined
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Project description:Purpose: Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest to enhance recombinant cell lines. Until recently, this focus has been challenged by a lack of efficacious, high throughput, and low-cost gene editing modalities and screening methods. In this work, we demonstrate an improved method for gene editing in CHO cells using CRISPR RNPs and characterize the endpoints of Cas9 and ZFN mediated genetic engineering. Methods: Amplicons were first assayed for quality using an Invitrogen Quant-iT dsDNA (Thermo, Cat Q33120) assay and gel electrophoresis to determine DNA concentration and DNA quality. Samples were then used for Sanger sequence based decomposition or processed downstream using NGS. For NGS, libraries were generated using the Illumina TruSeq Nano DNA kit (Illumina, Cat: 20015964). The libraries were sequenced using the Illumina MiSeq platform, with read length of 2x150bp. 4.5Gb of sequencing data was generated per DNA sample. Results: There was a tendency for decomposition to underestimate editing as compared to NGS as the indel size became larger. Despite these results, sequence decomposition was able to reproduce next-generation data in the context of gene editing efficiency across a short amplicon. Conclusions: We took advantage of the differential Indel spectrums induced by both ZFN and CRISPR to validate metrics for assess gene editing and show how deconvolution-based methods, can be utilized in the context of cell line development.
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Project description:Chinese Hamster Ovary (CHO) cells are frequently used to produce recombinant HIV envelope protein gp120. In this study, we characterized the CHO proteins that become co-purified with gp120 when lectin affinity chromatography is used. Many of co-purified CHO proteins identified were extracellular matrix components. We verified a method for separating gp120 from these CHO proteins with anion exchange chromatography, and assessed the biochemical activity of gp120 preparations with and without co-purified CHO proteins.
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Project description:We investigated CHO signaling events in a comparative expression and phospho proteomics approach of recombinant mAb producing XL99 cell line and according parental cells. First, on proteomic level differences between producer and parental cells were evaluated by label-free quantification of proteins at exponential growth. Next, a SILAC-based phosphoproteomic investigation was used to elucidate differential phosphorylation events following IGF treatment as well as differences between two producer cell lines and the parental cells.
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Project description:The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels, and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival pathways such as the phosphoinositide-3-kinase (PI3K/AKT), Mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT) and RAP1 pathways was observed, offering novel insights into how cell surface receptors and ligand mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/ survival-receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior.
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