Abstract
Simple SummaryResearch in large animal models has been hampered by the complexity to introduce new gene alterations, but this has been simplified by the discovery of the CRISPR/Cas system. Here, we have cloned a Cas9 minipig to generate a porcine model for pre-clinical research. Six viable piglets were produced and backcrossed to Göttingen minipigs for two generations. Primary cells from different organs were isolated, and multiple gene alterations were performed by CRISPR in vitro. In vivo activation of the Cas9 expression was conducted by viral delivery of the FlpO expression to the skin. Overall, we successfully cloned a Cas9-expressing minipig and confirmed gene alterations introduced by the CRISPR/Cas system to porcine cells.The generation of large transgenic animals is impeded by complex cloning, long maturation and gastrulation times. An introduction of multiple gene alterations increases the complexity. We have cloned a transgenic Cas9 minipig to introduce multiple mutations by CRISPR in somatic cells. Transgenic Cas9 pigs were generated by somatic cell nuclear transfer and were backcrossed to Göttingen Minipigs for two generations. Cas9 expression was controlled by FlpO-mediated recombination and was visualized by translation from red to yellow fluorescent protein. In vitro analyses in primary fibroblasts, keratinocytes and lung epithelial cells confirmed the genetic alterations executed by the viral delivery of single guide RNAs (sgRNA) to the target cells. Moreover, multiple gene alterations could be introduced simultaneously in a cell by viral delivery of sgRNAs. Cells with loss of TP53, PTEN and gain-of-function mutation in KRASG12D showed increased proliferation, confirming a transformation of the primary cells. An in vivo activation of Cas9 expression could be induced by viral delivery to the skin. Overall, we have generated a minipig with conditional expression of Cas9, where multiple gene alterations can be introduced to somatic cells by viral delivery of sgRNA. The development of a transgenic Cas9 minipig facilitates the creation of complex pre-clinical models for cancer research.
Highlights
Breakthroughs in regards to diagnosis and treatment of diseases, including cancer, have often been obtained from studies in small animal models
The SKT plasmid was generated by replacing the PTEN and TP53 single guide RNAs (sgRNA) with a STK11 sgRNA that was inserted into the pSpCas9(BB)-2A-GFP (PX458) plasmid (Addgene no. 48138) using the NotI and ClaI restriction sites
The guide efficacy was determined with the TIDE (Tracking of insertion and deletion (Indel) by Decomposition) software through transfection of porcine fibroblasts with the pSpCas9(BB)-2A-GFP plasmid (Addgene ID: 48138), harbouring the designated sgRNA followed by FACS of GFP positive cells
Summary
Breakthroughs in regards to diagnosis and treatment of diseases, including cancer, have often been obtained from studies in small animal models. Porcine cancer models have been generated with limited success, compared to the large number of mouse models [7,8,9,10,11,12,13,14]. The few successful cancer models in pigs have revealed new aspects of cancer biology, for example highlighted by the insight to TP53, which is not evolutionary conserved to the mouse [15]. Transgenic porcine disease models are generated by expensive and time-consuming methods such as pronuclear injection or somatic cell nuclear transfer (SCNT). Applying the CRISPR/Cas system to large animal models, such as pigs, could overcome some of the obstacles with generating new lines and study multiple gene alterations in vivo and in vitro. In vivo activation of Cas expression was performed in the skin, validating the design of the construct in vitro and in vivo
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