Si Nga Sou, Dirk-Jan Kleinjan, Susan J. Rosser Institute of Quantitative Biology, Biochemistry, and Biotechnology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FF, UK Correspondence: Si Nga Sou (si.sou@ed.ac.uk) Background 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 a CRISPR-dCas based synthetic transcription factors (sTF) system that modulates expression of endogenous mRNA and miRNA targets involved in protein transport and glycosylation. Materials and methods sTF utilises two forms of Cas9 proteins: Endonuclease inactive ‘dead’ Cas9 (dCas9) with trans-activator domain (VPR) attached and native cutting Cas9 (Fig. In Herceptin® expressing CHO-K1, we transiently expressed dCas9-VPR with sgRNAs against upstream of protein transport-related gene promoters (Napg, Rab5A & Aprc1b) for transcriptional activation, or transfecting dCas9 with sgRNAs against their promoter regions for suppression (Vamp4).

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To lower galactosyltransferase (β1,4-GalT)-associated miRNA expression (cgr-miR-181d-5p, cgr-miR500 & cgr-miR501-5p), CHO cells were co-expressed with dCas9 and sgRNAs against miRNA promoters; or with native Cas9 and sgRNAs against mature miRNA sequences [1]. MRNA and miRNA levels of target genes were quantified by q-rt-PCR, protein level of β1,4-GalTs by western blot, and secreted IgG yield by IgG-ELISA. Results The dCas9 approach receives up to 60% increase in IgG expression, along with 1.2 to 2.5-fold rise in Napg, Rab5A and Aprc1b mRNA levels. While repressing Vamp4 transcription leads to a negative effect on IgG yield (Fig. Our results show positive correlation between pathways involved in protein transport and recycling, and recombinant protein (rProtein) yield. Both Cas9 and dCas9 approaches reduce miR-181d-5p, miR500 & miR501-5p by around 35-50%, this simultaneously enhances β1,4-GalT1 & 4 expression by up to 2-fold, which could be useful in future engineering of rProtein glycosylation profiles for specific function. This system also provides a platform for concurrent manipulation of multiple mRNA and miRNA with dCas9, where dCas9 expression can be further controlled via AID- or ecDFR-Degron technology [2].

Conclusions Our works here present the potential of the CRISPRa/i system to easily reengineer or to study CHO cell metabolic pathways for more efficient rProtein production. The chemical inducible Cas9/dCas9 protein expression offers further control over multiple endogenous gene manipulation. Acknowledgements Authors thankfully acknowledge the Biotechnology and Biological Sciences Research Council for funding this research work. SNS thanks ESACT 2017 for providing her with the opportunity to present her work at the meeting.

Ccnp route ppt slides. Router B will accept this routing update, and realize that the interface receiving the update (Serial0) belongs to the same major network as the route entry of 10.1.0.0. It will then apply the subnet mask of its Serial0 interface to this route entry. We’ll assume that IGRP is configured correctly on all routers.

References 1. Chang H, Yi B, Ma R, Zhang X, Zhao H, Xi Y. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Scientific Reports 2016. Kleinjan D, Wardrope C, Sou S, Rosser S. A Toolkit of Tunable, Degron-tagged dCas9/Cpf1 Effectors for Multi-directional Drug-inducible control of Synthetic Gene Regulation.

Type Production Years Type Production Years Type Production Years Type Production Years 4X90 2001 - AC1 1985 AD1 1984 DWS4 1997 LPA2 1988 LPA3 1988 - LPW2 1988 LPW4 1988 LPWS2 1988 LPWS3 1988 LPWS4 1989 LPWT4 1996 - LPW3 1988 LT1 1974 LV1 1983 TR1 1985 TR2 1985 TR3 1985 TS1 1983 TS2 1983 TS3 1983 - TX2 1987 - 2001 TX3 1987 Lister Engines - Obsolete Models. Lister engine serial numbers. Lister Petter Engines - Current Models.