ROSA26-Cas9 transgenic pigs: a tool for in vivo genome editing

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We are modelling human cancers in pigs to aid development of new diagnostic procedures and treatments. CRISPR/Cas9 gene editing has dramatically simplified gene inactivation in large mammals. A particularly powerful strategy has recently been reported based on transgenic mice that express Cas9. This enables gene inactivation in chosen somatic cells by introducing guide RNAs targeted to genes of interest. Such somatic cell engineering has many uses, including mimicking spontaneous mutations responsible for particular cancers. We propose extending this technology to pigs by generating Cas9 transgenic pig lines. These would significantly enhance the power and scope of cancer modelling by greatly increasing the number of genes that can be investigated and reducing the time necessary. Importantly it would reduce the number of animals needed, in accordance with the principle of replacement, reduction and refinement for work with experimental animals. The work proposed makes use of three genetically modified pig lines we have already generated. We previously identified the porcine ROSA26 locus and used it to place a ubiquitously expressed dual fluorescent reporter of Cre recombination. We have also generated pigs with gene-targeted mutations in key cancer-related genes, including the APC1311 mutation (orthologous to human APC1309), which initiates colorectal polyposis, and a Cre-inducible form of oncogenic KRASG12D an important driver of colorectal and other cancersWe will generate constitutive and Cre-inducible Cas9 transgenes, place them by gene targeting at ROSA26 and make pigs by nuclear transfer. The cells used will carry the APC1311 mutation, so founder animals will develop polyps. Proof-of-principle studies will focus on constitutive Cas9. Guide RNAs targeted to cancer-related genes will be introduced endoscopically into polyps in the distal gut. Follow-up colonoscopies will monitor the phenotype and collect samples for molecular and immunohistological analyses.While founder animals are being generated we will determine the best method of delivering nucleic acids into polyps in vivo (e.g. electroporation, AAV vectors) by introducing Cre into polyps in APC1311 animals that also carry the fluorescent Cre reporter. The proportion of Cre-recombined cells visualised by fluorescence microscopy of biopsy samples will provide a measure of the success of transfection/transduction methods. If time allows, we will test local and cell-type specific activation of Cre-inducible Cas9 in vivo, using for example the VIL1 (villin) promoter. Our future plans are to combine Cre-inducible Cas9 with Cre-inducible KRASG12D to investigate oncogenic KRAS expression with inactivation of other cancer-related genes. This will include selective inactivation of KRAS effectors to analyse signalling pathways important for colorectal cancer. The Cas9 pigs will be a useful resource for many researchers, enabling genome engineering in pigs for a wide variety of fields.