Abstract 3706: The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues

Abstract

The aim of the project is to generate a toolbox pig model. This is being done because the pig probably is the best non-primate model in translational medicine. Unfortunately, pig models have proven difficult to produce. The two main complications have been the cloning of viable pigs and that just a few genes are altered, which often is not sufficient to induce the desired disease. Especially, models based on multiple genetic modifications of the pig genome, e.g. cancer, are challenging to generate. This was changed with the development of the CRISPR-Cas9 technology, which made it possible to perform multiple in vivo gene editing. We have cloned an inducible Cas9 expressing minipig and have obtained healthy F1 offspring. Activation of the Cas9 expression is attained by transduction with adeno-associated virus that delivers both the recombinase and the guide RNA to the designated tissue. This design provides spatial and temporal control of the activation. The Cas9 expression is mediated by a ubiquitous promotor, which also co-expresses fluorescent proteins for expression analysis. Transgenic Cas9 expression is observed in all major organs with strong expression in the epithelia, muscles and hepatocytes. Successful in vitro gene editing has been conducted in primary keratinocytes and fibroblast obtained from the transgenic pigs, which confirms the genetic design. By delivery of our construct by adeno-associated virus to the skin of the pigs, In vivo activation of the transgenic construct has been achieved. To validate the model, lung cancer has been induced in nine pigs by alteration a panel of cancer related genes including STK11, TP53, PTEN, and KRAS, all key drivers of human lung cancer. To obtain the constitutive active KRAS-G12D isoform of the KRAS oncogene a repair template was supplied. The pigs are being followed by PET/CT scanning in combination with 18F-FDG as a tracer. Preliminary data indicates abnormalities/cancer initiation in the pigs eight months after induction. In conclusion, an inducible Cas9 expressing minipig allows rapid gene editing to study different scenarios of human cancer in a pig model. The development of the Cas9 pig will extend the variety of pig disease models, both to generate a model of lung cancer as proposed but also models for other cancers. Citation Format: Martin F. Berthelsen, Maria Riedel, Mikkel H. Vendelbo, Aage K. Alstrup, Frederik Dagnaes-Hansen, Yonglun Luo, Simone S. Moller, Henrik Callesen, Jannik E. Jakobsen, Martin K. Thomsen. The CRISPR-Cas9 minipig: A transgenic toolbox pig to produce specific genome editing in designated tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3706.