The CRISPR/Cas9 Minipig—A Transgenic Minipig to Produce Specific Mutations in Designated Tissues

Abstract

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.

In Vivo Application of CRISPR/Cas9 Revealed Implication of Foxa1 and Foxp1 in Prostate Cancer Proliferation and Epithelial Plasticity

“…MEF cells were transfected with X-tremeGENE-9 according to the manufacturer’s protocols and puromycine selections were applied for 48 h (2 µg/mL). AAV production was conducted in HEK293T cells as described before [ 22 ].…”

Characterization of the immune system of Ellegaard Göttingen Minipigs - An important large animal model in experimental medicine

“…A huge advantage over rodents is that long-term studies can be conducted, which is essential for many questions in experimental medicine and study of disease courses. Further, genetically modified EGMs with adaptations that resemble genetic alterations responsible for human diseases have been generated ( 16 , 17 ). All this makes EGMs an interesting model, however, knowledge about their immune system is still scarce.…”

Application of CRISPR for In Vivo Mouse Cancer Studies

“…The discovery of CRISPR/Cas9 has changed the way to model human cancer in vitro and in vivo. The application of CRISPR/Cas9 to study cancer in vivo will continue to develop and be applied to different species, not restricted to the mouse [ 28 , 62 , 63 ]. CRISPR/Cas9 has mainly been used to study loss-of-function mutations of potential tumor suppressor genes.…”

“…A plasmid delivery to other cells has very low efficiency but combined with electroporation, it can be increased. By electroporation, gene editing has been done in astrocytes, keratinocytes, muscles, and pancreatic cells [ 25 , 26 , 27 , 28 ]. The advantage of plasmid delivery is the ability to deliver a large cargo, as the coding sequence for Cas9 protein is 4.1 kbp.…”