A pooled CRISPR/AsCpf1 screen using paired gRNAs to induce genomic deletions in Chinese hamster ovary cells

Valerie Schmieder, Neža Novak, Heena Dhiman, Ly Ngoc Nguyen, Evgenija Serafimova, Gerald Klanert, Martina Baumann, Helene Faustrup Kildegaard, Nicole Borth

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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.
Original languageEnglish
Article numbere00649
Journal Biotechnology Reports
Volume31
Number of pages10
ISSN2215-017X
DOIs
Publication statusPublished - 2021

Keywords

  • Genetic screen
  • CRISPR/AsCpf1
  • Paired gRNAs
  • Genomic deletion
  • Chinese hamster ovary cells

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