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• W3176774265 endingPage "e00649" @default.
• W3176774265 startingPage "e00649" @default.
• W3176774265 abstract "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." @default.
• W3176774265 created "2021-07-05" @default.
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• W3176774265 date "2021-09-01" @default.
• W3176774265 modified "2023-10-05" @default.
• W3176774265 title "A pooled CRISPR/AsCpf1 screen using paired gRNAs to induce genomic deletions in Chinese hamster ovary cells" @default.
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• W3176774265 doi "https://doi.org/10.1016/j.btre.2021.e00649" @default.
• W3176774265 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/8261548" @default.
• W3176774265 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/34277363" @default.
• W3176774265 hasPublicationYear "2021" @default.
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