CRISPR-Cas Based Synthetic Transcription Factors: A Strategy for Improving Bioproduction in CHO Cells

CRISPR-Cas9, synthetic transcription factor, miRNA/mRNA expression, productivity, glycosyltransferase

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Despite advances in Chinese hamster ovary (CHO) cell bioprocess optimisation, production of large complex proteins remains costly and high degree of variability among final products is problematic. Novel strategies that target molecular pathways for high product yield and consistency are vital. To overcome this bottleneck, we developed CRISPR-Cas based synthetic transcription factors (sTF) that modulate expression of endogenous mRNA and miRNA targets involved in protein trafficking, gene transcription and glycosylation.

sTF utilised two forms of Cas9 proteins: Endonuclease inactive ‘dead’ Cas9 (dCas9) with activator attached and native genome editing Cas9. In EGFP expressing CHO-K1, we transiently expressed dCas9 with sgRNAs against upstream of protein transport-related gene promoters (Rab5A & Aprc1b) and gene transcription (RasL11A) for transcriptional activation, or against their promoter regions for suppression. To lower galactosyltransferase (GalT) associated miRNAs expression (miR-181d-5p, miR500 & miR501), cells were co-expressed with dCas9 and sgRNAs against miRNA promoters; or with native Cas9 and sgRNAs against mature miRNA sequences. In all cases, mRNA and miRNA levels of target genes were quantified by q-rt-PCR, protein expression of GalTs and EGFP by western blot.

The dCas9 approach receives up to 30% increase in EGFP expression, along with 2 to 5-fold rise in Rab5A, Aprc1b and RasL11A mRNA levels. Our results show positive correlation between protein trafficking and rProtein yield. Both Cas9 and dCas9 approaches reduce miR-181d-5p, miR500 & miR501 by around 35%, this simultaneously enhances GalT1 & 4 expression by up to 2-fold, which will be advantageous towards protein glycosylation. This system allows concurrent manipulation of multiple mRNA and miRNA with dCas9, where dCas9 expression can be further controlled via AID- or ecDFR-Degron technology. This technology is an useful tool to create in vitro circuits during CHO cell engineering to specifically improve bioproduction.

Reference

[1].Chang H et al. 2016. Sci. Rep. 6:22312