ROSA26-Cas9 transgenic pigs: a tool for in vivo genome editing
Final Report Abstract
The main project goal was to generate Cas9 transgenic pig for 'somatic cell engineering'. Such a pig would have many valuable uses, including mimicking spontaneous mutations responsible for particular cancers. These animals have been successfully generated, the pigs are healthy, fertile and transgene inheritance follows mendelian rules. Most importantly, they express the Cas9 gene in all organs analysed, and support both non-homologous end-joining and homologous mediated repair upon addition of guide RNAs. Genome editing has been shown for several different endogenous targets. F1 animals have been obtained that carry both the Cas9 and a conditional oncogenic KRASG12D/+ mutation. Other crosses with APC1311 and to obtain homozygous Cas9 animals are underway, with births of piglets expected in late November and December 2019. The Cas9 animal will now enable the generation of new cancer models. By necessity most genetically modified cancer models, mainly in mice, have so far been based on a small number of mutant genes. This does not reflect the genetic complexity of spontaneous human cancers. Local administration of sets of guide RNAs to the Cas9 pigs will enable several genes to be edited simultaneously, and the combined effects analysed. Vectors containing multiple guide RNAs for several tumour suppressor genes (SMAD4, TP53, p16, BRACA2 for pancreatic cancer and SMAD4, TP53, p16, PTEN, APC for colorectal cancer) have already been tested in cell culture and are now being assessed in vivo. Somatic genome editing can be applied to the study of different cancers but also to many other diseases with a genetic component. For example, we have already been asked to provide Cas9 animals for cardiovascular projects and it is planned to use these animals within the consortia SFB 1321, SFB 1371 and TRR 267. The Cas9 pig lends itself to both somatic and germline genome editing, it provides a new tool for reverse genetics in livestock and enables the modification of single and multi-loci traits. Beyond biomedical applications, traits that improve animal health and disease resistance are of particular interest. We are confident that the Cas9 animals will be a very useful resource for many other researchers, enabling genome engineering in pigs for a wide variety of fields.
