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

• Published in: Biotechnology reports (Amsterdam, Netherlands) Vol. 31; p. e00649
• Main Authors: Schmieder, Valerie, Novak, Neža, Dhiman, Heena, Nguyen, Ly Ngoc, Serafimova, Evgenija, Klanert, Gerald, Baumann, Martina, Kildegaard, Helene Faustrup, Borth, Nicole
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2021; Elsevier
• Subjects: Chinese hamster ovary cells; CRISPR/AsCpf1; Genetic screen; genomic deletion; paired gRNAs; CRISPR/AsCpf1; CPM; DE; lncRNA; UP-TSS; CHO; FACS; DR; gRNA; Chinese hamster ovary cells; FDR; DOWN-TTS; oligo; CRISPR; EpoFc; ncGene; paired gRNAs; dCas9; GS; Genetic screen; PCA; EV; pgRNA; AsCpf1; sgRNA; TMM; µ; VCD; genomic deletion; Cpf1; Cas9; NGS; RMCE; NTC; FC; PAM; Qp
• ISSN: 2215-017X; 2215-017X
• DOI: 10.1016/j.btre.2021.e00649

• Development of a small-scale CRISPR/AsCpf1 screen in CHO.• Usage of paired gRNAs enables full deletion of coding or noncoding genomic regions.• Growth perturbing paired gRNAs identified.• Key points for considerations in future screens identified. 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. Graphical workflow for a CRISPR/AsCpf1 paired gRNA (pgRNA) genomic deletion screen in Chinese hamster ovary (CHO) cells. pgRNA library ordered as a single-stranded oligonucleotide pool, followed by PCR amplification and cloning into the delivery plasmid backbone. Next, stably pgRNA expressing pre-screening cell pools were generated. By the application of the AsCpf1 endonuclease, genomic deletions were induced, and cell pools were screened for the desired phenotype. In parallel, pre-screening cell pools were treated with Cas9 resulting in no alteration of the genome. Comparison of pgRNA abundance between AsCpf1 and Cas9 treated samples after screening procedure identified phenotype modifying hits. Critical pooled CRISPR screening steps are marked by exclamation marks. [Display omitted]