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

doi:10.1016/j.btre.2021.e00649

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

Biotechnol Rep (Amst). 2021 Jun 20:31:e00649. doi: 10.1016/j.btre.2021.e00649. eCollection 2021 Sep.

Authors

Valerie Schmieder^{1

2}, Neža Novak^{1

2}, Heena Dhiman^{1

2}, Ly Ngoc Nguyen^{1

2}, Evgenija Serafimova^{1

2}, Gerald Klanert², Martina Baumann², Helene Faustrup Kildegaard³, Nicole Borth^{1

2}

Affiliations

¹ Department of Biotechnology, BOKU University of Natural Resources and Life Sciences, Muthgasse 18, Vienna, Austria.
² acib GmbH, Austrian Centre of Industrial Biotechnology, Muthgasse 11, Vienna, Austria.
³ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Building 220, Kemitorvet, Kgs. Lyngby, Denmark.

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.

Keywords: AsCpf1, Cpf1 from Acidaminococcus sp BV3L6; CHO, Chinese hamster ovary; CPM, counts per million reads mapped; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR/AsCpf1; Cas9, CRISPR-associated protein 9; Chinese hamster ovary cells; Cpf1, CRISPR-associated protein in Prevotella and Francisella; DE, differentially expressed; DOWN-TTS, downstream transcription termination site; DR, differentially represented; EV, empty vector; EpoFc, Erythropoietin Fc fusion protein; FACS, fluorescence activated cell sorting; FC, fold change; FDR, false discovery rate; GS, glutamine synthetase; Genetic screen; NGS, next generation sequencing; NTC, no template control; PAM, protospacer adjacent motif; PCA, principal component analysis; Qp, specific productivity; RMCE, recombinase-mediated cassette exchange; TMM, trimmed mean of M values; UP-TSS, upstream transcription start site; VCD, viable cell density; dCas9, deactivated Cas9; gRNA, guide RNA; genomic deletion; lncRNA, long non-coding RNA; ncGene, non-coding gene; oligo, oligonucleotide; paired gRNAs; pgRNA, paired gRNA; sgRNA, single guide RNA; µ, growth rate.