Animal models of human diseases play a critical role in medical research. Pigs are anatomically and physiologically more like humans than are small rodents such as mice, making pigs an attractive option for modeling human diseases. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, and various genetic diseases. We also discuss areas that need to be improved. Animal models of human diseases play a critical role in medical research. Advances in recent years in genetic engineering have facilitated the rapid rise of pig models for use in studies of human disease. In the present review, we summarize the current status of pig models for human cardiovascular, metabolic, neurodegenerative, various genetic diseases and xenotransplantation.

Significance: Because genome-wide CRISPR/Cas9 libraries are mostly constructed in lentiviral vectors, direct in vivo screening has not been possible as a result of low efficiency in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Through hydrodynamic tail vein injections, we delivered a PB-CRISPR library into mouse liver. Rapid tumor formation could be observed in less than 2 mo. By sequencing analysis of PB-mediated gRNA insertions, we identified corresponding genes mediating tumorigenesis. Our results demonstrate that PB is a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries for phenotype-driven screens.

To date no authentic embryonic stem cell (ESC) line or germline-competent-induced pluripotent stem cell (iPSC) line has been established for large animals. Despite this fact, there is an impression in the field that large animal ESCs or iPSCs are as good as mouse counterparts. Clarification of this issue is important for a healthy advancement of the stem cell field. Elucidation of the causes of this failure in obtaining high quality iPSCs/ESCs may offer essential clues for eventual establishment of authentic ESCs for large animals including humans. To this end, we first generated porcine iPSCs using nonintegrating replicating episomal plasmids. Although these porcine iPSCs met most pluripotency criteria, they could neither generate cloned piglets through nuclear transfer, nor contribute to later stage chimeras through morula injections or aggregations. We found that the reprogramming genes in iPSCs could not be removed even under negative selection, indicating they are required to maintain self-renewal. The persistent expression of these genes in porcine iPSCs in turn caused differentiation defects in vivo. Therefore, incomplete reprogramming manifested by a reliance on sustained expression of exogenous-reprogramming factors appears to be the main reason for the inability of porcine iPSCs to form iPSC-derived piglets. Stem Cells 2015;33:3228–3238

Efficient exogenous DNA integration can be mediated by Cas9 through the non-homology end-joining pathway. However, such integrations are often imprecise and contain a variety of mutations at the junctions between the external DNA and the genomic loci. Here we describe a microhomology-dependent targeted integration method, designated MITI, for precise site-specific gene insertions. We found that the MITI strategy yielded higher knock-in accuracy than Cas9 HITI for the insertion of external DNA and tagging endogenous genes. Furthermore, in combination with negative selection and four different CrRNAs targeting donor vectors and genome-targeted sites with a CrRNA array, MITI facilitated precise ligation at all junctions. Therefore, our Cas12a-based MITI method increases the repertoire of precision genome engineering approaches and provides a useful tool for various gene editing applications.