CRISPR/Cas9 mediated targeting of microRNA-24 improves the bioprocess phenotype of Chinese Hamster Ovary cells

Chinese Hamster Ovary (CHO) cells are the prominent cell line used in biopharmaceutical production. Although optimisation efforts have led to a vast increase in productivity, CHO cells yield less than other expression systems like yeast or bacteria. To improve yields and find beneficial bioprocess phenotypes, genetic engineering plays an essential role in recent research. The miR-23 cluster with its genomic paralogues (miR-23a and miR-23b) was first identified as differentially expressed during temperature shift, suggesting its role in proliferation and productivity. The common approach to deplete miRNAs is the use of a sponge decoy which, requires the introduction of reporter genes. As an alternative this work aims to knockdown miRNA expression using the recently developed CRISPR/Cas9 system which does not require a reporter transcript. This system consists of two main components: the single guide RNA (sgRNA) and an endonuclease (Cas9) which induces double strand breaks (DSBs). These DSBs can result in insertion or deletion (indels) of base pairs which can disrupt miRNA function and processing. A CHO-K1 cell line stably expressing an IgG was used for knockdown experiments. SgRNAs were designed to target the seed region of miR-24-3p and stable mixed populations were generated. It was shown that miRNA expression for miR-24-3p as well as miR-24-5p was significantly reduced in mixed populations. Please click Additional Files below to see the full abstract