ena-STUDY-UNIVERSITY OF NATURAL RESOURCES AND LIFE SCIENCES,-17-12-2019-18:09:01:691-1405
DNA sequences of paired gRNA integration site from a CRISPR/AsCpf1 deletion screen in CHO
Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins and tremendous progress has been made in the past few years in terms of sequence and annotation quality Nevertheless, the link between genotype and phenotype is still not fully understood. Most studies are mainly focused on protein-coding genes, which only make up around 3% of the mammalian genome. However, untranslated genes like long-non-coding RNA (lncRNA) also play an important role in the regulation of cellular processes that can have an enormous impact on the phenotype of cells. In this study, the first CRISPR-mediated screening approach that uses full deletion of genomic regions is presented for CHO cells.An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was successfully established and used to generate a library of 2,348 pgRNA sequences tackling differentially expressed lncRNA in CHO. These were synthesized, cloned into a plasmid and stably integrated into CHO using recombinase-mediated cassette exchange. AsCpf1 was delivered to induce genomic deletions and Cas9 was used in parallel as a negative control. Cells were cultivated over two sequential batches to deplete pgRNAs which were negatively affecting growth. Next generation sequencing (NGS) analysis of the plasmid pool revealed sufficient, but highly variable representation of pgRNAs. By comparing pgRNA abundance in AsCpf1 and Cas9 samples, depleted candidates were identified and further validated for their effect on growth in batch cultivations. Critical steps and potential caveats during the preparation of CRISPR deletion screens in CHO were identified. The here presented screening strategy is the first of its sort in CHO cells and was demonstrated for a small-scale deletion approach. Lessons learned now open the opportunity to study the entire genome.
Chinese hamster ovary (CHO) cells are the most widely used host for the expression of therapeutic proteins and tremendous progress has been made in the past few years in terms of sequence and annotation quality Nevertheless, the link between genotype and phenotype is still not fully understood. Most studies are mainly focused on protein-coding genes, which only make up around 3% of the mammalian genome. However, untranslated genes like long-non-coding RNA (lncRNA) also play an important role in the regulation of cellular processes that can have an enormous impact on the phenotype of cells. In this study, the first CRISPR-mediated screening approach that uses full deletion of genomic regions is presented for CHO cells.An in silico pipeline for the computational high-throughput design of paired guide RNAs (pgRNAs) directing CRISPR/AsCpf1 was successfully established and used to generate a library of 2,348 pgRNA sequences tackling differentially expressed lncRNA in CHO. These were synthesized, cloned into a plasmid and stably integrated into CHO using recombinase-mediated cassette exchange. AsCpf1 was delivered to induce genomic deletions and Cas9 was used in parallel as a negative control. Cells were cultivated over two sequential batches to deplete pgRNAs which were negatively affecting growth. Next generation sequencing (NGS) analysis of the plasmid pool revealed sufficient, but highly variable representation of pgRNAs. By comparing pgRNA abundance in AsCpf1 and Cas9 samples, depleted candidates were identified and further validated for their effect on growth in batch cultivations. Critical steps and potential caveats during the preparation of CRISPR deletion screens in CHO were identified. The here presented screening strategy is the first of its sort in CHO cells and was demonstrated for a small-scale deletion approach. Lessons learned now open the opportunity to study the entire genome.