Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications.

A B S T R A C TThe coronaviruses, porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine deltacoronavirus (PDCoV) represent important sources of neonatal diarrhea on pig farms. The requirement for aminopeptidase N (APN) as a receptor for TGEV, but not for PEDV, is well established. In this study, the biological relevance of APN as a receptor for PDCoV was tested by using CRISPR/Cas9 to knockout the APN gene, ANPEP, in pigs. Porcine alveolar macrophages (PAMs) from ANPEP knockout (KO) pigs showed resistance to PDCoV infection. However, lung fibroblast-like cells, derived from the ANPEP KO PAM cultures, supported PDCoV infection to high levels. The results suggest that APN is a receptor for PDCoV in PAMs but is not necessary for infection of lung-derived fibroblast cells. The infection of the ANPEP KO pigs with PDCoV further confirmed that APN is dispensable as a receptor for PDCoV.

During the past 50 years, physicians have become increasingly dissatisfied with certain aspects of their profession. Dissatisfaction has intensified with the advent of managed care in the late 20th century, the medical liability crisis, and the growing divergence between the professional and personal expectations placed upon physicians and their practical ability to meet these expectations. These and other factors have encroached on physician autonomy, the formerly ascendant professional value within medicine. As the underlying values and practical realities of the broader American health care system have changed, the professional values and practices of physicians have failed to adapt correspondingly, resulting in a "professionalism gap" that contributes to physician dissatisfaction. To improve the outlook and efficacy of modern American physicians, the profession must adopt a new values framework that conforms to today's health care system. This means foregoing the 20th century's preferred "independent physician" model in favor of a new professional structure based on teamwork and collaboration. Convincing established physicians to embrace such a model will be difficult, but opportunities exist for significant progress among a new generation of physicians accustomed to the realities of managed care, flexible practice models, and health information technology. The teaching of clinical anatomy, given its incorporation of student collaboration at the earliest stages of medical education, offers a prime opportunity to introduce this generation to a reinvigorated code of professionalism that should reduce physician dissatisfaction and benefit society.

BackgroundRecent advancements in gene editing techniques have increased in number and utility. These techniques are an attractive alternative to conventional gene targeting methods via homologous recombination due to the ease of use and the high efficiency of gene editing. We have previously produced cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) knockout (KO) pigs in a Minnesota miniature pig genetic background. These pigs were generated using zinc-finger nucleases (ZFNs) in combination with donor DNA containing a total homology length of 1600 bp (800-bp homology on each arm). Our next aim was to introduce the targeted disruption of alpha-1,3-galactosyltransferase (GGTA1) in the CMAH KO genetic background and evaluate the effect of donor DNA homology length on meganuclease-mediated gene targeting.MethodsZinc-finger nucleases from a previous CMAH KO experiment were used as a proof of concept to identify a correlation between the length of donor DNA homology and targeting efficiency. Based on those results, experiments were designed to use transcription activator-like effector nucleases (TALENs) to generate bi-allelically modified GGTA1 cells using donor DNAs carrying various lengths of homology. Donor DNA was designed to symmetrically flank the predicted cleavage sites in CMAH and GGTA1 for both ZFN and TALEN cleavage sites, respectively. For both genes, the length of total homology ranged from 60 to 1799 bp. Sialyltransferase gene expression profiles were evaluated in CMAH and GGTA1 double KO pig cells and were compared to wild-type and CMAH KO cells.ResultsIntroduction of donor DNA with ZFNs demonstrated that small amounts of homology (60 bp) could facilitate homology-directed repair during ZFN-mediated targeting of CMAH; however, donor DNA with longer amounts of homology resulted in a higher frequency of homology-directed repair. For the GGTA1 KO experiments that used TALENs and donor DNA, donor DNA alone did not result in detectable bi-allelic conversion of GGTA1. As the length of donor DNA increased, the bi-allelic disruption of GGTA1 increased from 0.5% (TALENs alone, no donor DNA present) to a maximum of 3% (TALENs and donor DNA with total homology of 1799 bp). Inclusion of homologous donor DNA in TALEN-mediated gene targeting facilitated a higher incidence of bi-allelically modified cells. Using the generated cells, we were able to demonstrate the lack of GGTA1 expression and the decrease in gene expression sialyltransferase-related genes.ConclusionsThe approach of using donor DNA in conjunction with a meganuclease can be used to increase the efficiency of gene targeting. The gene editing methods can be applied to other genes as well as other mammalian systems. Additionally, gene expression analysis further confirms that the CMAH/GGTA1 double KO pigs can be a valuable source for the study of pig-to-human xenotransplantation.

Although several techniques have been developed to create gene knockouts in pigs, homologous recombination will continue to be required for site-specific genome modifications that are more sophisticated than gene disruption (base changes, domain exchanges, conditional knockouts). The objective of the present paper was to improve the efficiency of homologous recombination in porcine fetal fibroblasts, which would be used to produce gene knockout pigs by somatic cell nuclear transfer. A promoter-trap was used to enable selection of GGTA1 targeted cells. Cells were transfected with either a single stranded or double stranded targeting vector, or a vector, with or without a negative selectable marker gene (diphtheria toxin-A). Although targeting efficiencies were numerically lower for single stranded targeting vectors, statistical differences could not be detected. Similarly, the use of a negative selectable marker (in cis or trans) provided numerically lower targeting efficiencies, statistical differences again could not be detected. Overall, the targeting efficiencies ranged from 1.5×10−5 to 2.5×10−6 targeting events per transfected cell. Given the results, it may be applicable to investigate multiple enrichment techniques for homologous recombination, given that every targeted locus is different.