Genetically modified pigs play an important role in agriculture and biomedical research; hence, new efficient methods are needed to obtain genetically engineered cells and animals. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated) system represents an effective genome editing tool. It consists of two key molecules: single guide RNA (sgRNA) and the Cas9 endonuclease that can be introduced into the cells as one plasmid. Typical delivery methods for CRISPR/Cas9 components are limited by low transfection efficiency or toxic effects on cells. Here, we describe the use of magnetic nanoparticles and gradient magnetic field to improve delivery of CRISPR/Cas9 constructs into porcine fetal fibroblasts. Polyethylenimine-coated nanoparticles with magnetic iron oxide core were used to form magnetic plasmid DNA lipoplexes. CRISPR/Cas9 construct was prepared to induce site-specific cutting at the porcine H11 locus. Quantitative assessment of genomic cleavage by sequence trace decomposition demonstrated that the magnetofection efficiency was more than 3.5 times higher compared to the classic lipofection method. The Tracking of Indels by Decomposition web tool precisely determined the spectrum of indels that occurred. Simultaneously, no additional cytotoxicity associated with the utilization of magnetic nanoparticles was observed. Our results indicate that magnetofection enables effective delivery of the CRISPR/Cas9 construct into porcine fetal fibroblasts with low cell toxicity.

The shortage of organs for transplantation is a well-known issue of modern medicine. The domestic pig has proved to be most suitable for xenotransplantation purposes. It is necessary to modify the pig genome to eliminate the immunological differences resulting from its phylogenetic remoteness from humans. We present a generation of pigs with human CD55 and ULBP1 gene knockout. Both modifications were introduced using the CRISPR/Cas9 system. A mixture of Cas9-D10A mRNA, a pair of sgRNAs and the pCD55 donor vector were introduced into the pronucleus of the fertilized porcine oocyte. After microinjection the three surrogates delivered a total of 13 piglets. The analysis showed four piglets with indels in the targeting site of exon 2 of the pULBP1 gene. All four pigs were altered in a biallelic manner, showing different sequences in each mutant allele. One piglet also showed one allele interrupted with a CD55-expressing cassette. The analyses confirmed the integration and the expression of the CD55 transgene in the targeted site. The human serum cytotoxicity test results showed that the highest viability of modified cells was 84.42% compared to the control. The cytometric analysis suggests that the CD55-expressing cassette was integrated with the genome in the transcription active site.

There are a number of human diseases, which can lead to organ failure. The consequence is often the need for a transplant. The number of performed operations is very low due to the shortage of organs for transplantation. As a consequence, the number of people waiting for transplant is still growing. The solution to this situation may be xenotransplantation. Xenotransplantation word comes from the Greek xenos meaning stranger, the other. It is defined as any procedure that involves the transplantation, implantation or infusion of tissues or zoonotic organs into a human recipient, but also human body fluids, cells, tissues, organs (or fragments) that have ex vivo contact with zoonotic cells, tissues or organs. One of the obstacles of the xenograft transplantation is the risk of animal pathogens transmission to the humans. Viruses that pose risk in the non-human primates-to-human xenotransplantation includes: the human immunodeficiency virus - HIV and the Marburg virus described in this paper. In addition viruses, which is a problem in pig-to-human xenotransplantation have also been described, including: porcine endogenous retrovirus - PERV, porcine cytomegalovirus - PCMV, porcine lymphotropic herpesvirus - PLHV and hepatitis E virus - E - HEV. This review of literature is the latest knowledge of the microbiological safety of xenotransplantation.

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Animals as a source of organs and tissues for xenotransplantation could become a backup solution for the growing shortage of human donors. The presence of human xenoreactive antibodies directed against Galα1,3Gal antigens on the cell surface of a pig donor triggers the activation of the complement leading to a hyperacute reaction. The development of genetic engineering techniques has enabled the modification of genomes by knocking in and/or knocking out genes. In this paper, we report the generation of modified pigs with ZFN mediated disruption of the GGTA1 gene encoding the enzyme responsible for synthesis of Galα1,3Gal antigens. ZFN plasmids designed to target the exon 9 region of the pig GGTA1 gene encoding the catalytic domain were injected into the pronuclei of fertilized egg cells. Among 107 piglets of the F0 generation analyzed, one female with 9-nt deletion in exon 9 of the GGTA1 gene was found. 13 of 33 piglets of the F1 generation represented the +/-GGTA1 genotype and 2 of 13 F2 piglets represented the -/-GGTA1 genotype. No changes in the animals' behavior, phenotype or karyotype were observed. Analysis confirmed heredity of the trait in all animals. A complex functional analysis of the modified animals, including flow cytometry, human serum cytotoxicity test and immunohistochemical detection, was performed to estimate the phenotype effect of genetic modification and this indicated an efficient GGTA1 knock-out in modified pigs.