Swine ANP32A Supports Avian Influenza Virus Polymerase.

Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as "mixing vessels," being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus "mixing vessels." In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.

Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 protein is expressed at high levels on the surface of specific macrophage types, and a soluble form is circulating in blood. CD163 has been described as a fusion receptor for PRRSV, with the scavenger receptor cysteine-rich domain 5 (SRCR5) region having been shown to be the interaction site for the virus. As reported previously, we have generated pigs in which exon 7 of the CD163 gene has been deleted using CRISPR/Cas9 editing in pig zygotes. These pigs express CD163 protein lacking SRCR5 (ΔSRCR5 CD163) and show no adverse effects when maintained under standard husbandry conditions. Not only was ΔSRCR5 CD163 detected on the surface of macrophage subsets, but the secreted, soluble protein can also be detected in the serum of the edited pigs, as shown here by a porcine soluble CD163-specific enzyme-linked immunosorbent assay (ELISA). Previous results showed that primary macrophage cells from ΔSRCR5 CD163 animals are resistant to PRRSV-1 subtype 1, 2, and 3 as well as PRRSV-2 infection in vitro Here, ΔSRCR5 pigs were challenged with a highly virulent PRRSV-1 subtype 2 strain. In contrast to the wild-type control group, ΔSRCR5 pigs showed no signs of infection and no viremia or antibody response indicative of a productive infection. Histopathological analysis of lung and lymph node tissue showed no presence of virus-replicating cells in either tissue. This shows that ΔSRCR5 pigs are fully resistant to infection by the virus.IMPORTANCE Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiological agent of PRRS, causing late-term abortions, stillbirths, and respiratory disease in pigs, incurring major economic losses to the worldwide pig industry. The virus is highly mutagenic and can be divided into two species, PRRSV-1 and PRRSV-2, each containing several subtypes. Current control strategies mainly involve biosecurity measures, depopulation, and vaccination. Vaccines are at best only partially protective against infection with heterologous subtypes and sublineages, and modified live vaccines have frequently been reported to revert to virulence. Here, we demonstrate that a genetic-control approach results in complete resistance to PRRSV infection in vivo CD163 is edited so as to remove the viral interaction domain while maintaining protein expression and biological function, averting any potential adverse effect associated with protein knockout. This research demonstrates a genetic-control approach with potential benefits in animal welfare as well as to the pork industry.

Precision engineering for PRRSV resistance in pigs: Macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a panzootic infectious disease of pigs, causing major economic losses to the world-wide pig industry. PRRS manifests differently in pigs of all ages but primarily causes late-term abortions and stillbirths in sows and respiratory disease in piglets. The causative agent of the disease is the positive-strand RNA PRRS virus (PRRSV). PRRSV has a narrow host cell tropism, limited to cells of the monocyte/macrophage lineage. CD163 has been described as a fusion receptor for PRRSV, whereby the scavenger receptor cysteine-rich domain 5 (SRCR5) region was shown to be an interaction site for the virus in vitro. CD163 is expressed at high levels on the surface of macrophages, particularly in the respiratory system. Here we describe the application of CRISPR/Cas9 to pig zygotes, resulting in the generation of pigs with a deletion of Exon 7 of the CD163 gene, encoding SRCR5. Deletion of SRCR5 showed no adverse effects in pigs maintained under standard husbandry conditions with normal growth rates and complete blood counts observed. Pulmonary alveolar macrophages (PAMs) and peripheral blood monocytes (PBMCs) were isolated from the animals and assessed in vitro. Both PAMs and macrophages obtained from PBMCs by CSF1 stimulation (PMMs) show the characteristic differentiation and cell surface marker expression of macrophages of the respective origin. Expression and correct folding of the SRCR5 deletion CD163 on the surface of macrophages and biological activity of the protein as hemoglobin-haptoglobin scavenger was confirmed. Challenge of both PAMs and PMMs with PRRSV genotype 1, subtypes 1, 2, and 3 and PMMs with PRRSV genotype 2 showed complete resistance to viral infections assessed by replication. Confocal microscopy revealed the absence of replication structures in the SRCR5 CD163 deletion macrophages, indicating an inhibition of infection prior to gene expression, i.e. at entry/fusion or unpacking stages.

Unexpected mutations were expected and unrelated to CRISPR-Cas9 activity.

The scientific journal Nature Methods have just retracted a publication that reported numerous unexpected mutations after a CRISPR-Cas9 experiment based on collecting whole genome sequencing information from one control and two experimental genome edited mice. In the intervening 10 months since publication the data presented have been strongly contested and criticized by the scientific and biotech communities, through publications, open science channels and social networks. The criticism focused on the animal used as control, which was derived from the same mouse strain as the experimental individuals but from an unrelated sub-colony, hence control and experimental mice were genetically divergent. The most plausible explanation for the vast majority of the reported unexpected mutations were the expected underlying genetic polymorphisms that normally accumulate in two different colonies of the same mouse strain which occur as a result of spontaneous mutations and genetic drift. Therefore, the reported mutations were most likely not related to CRISPR-Cas9 activity.

Animal and cellular models of human disease.

In this eighteenth (2016) Annual Review Issue of The Journal of Pathology, we present a collection of 19 invited review articles that cover different aspects of cellular and animal models of disease. These include genetically-engineered models, chemically-induced models, naturally-occurring models, and combinations thereof, with the focus on recent methodological and conceptual developments across a wide range of human diseases.

Engineering large animal models of human disease.

The recent development of gene editing tools and methodology for use in livestock enables the production of new animal disease models. These tools facilitate site-specific mutation of the genome, allowing animals carrying known human disease mutations to be produced. In this review, we describe the various gene editing tools and how they can be used for a range of large animal models of diseases. This genomic technology is in its infancy but the expectation is that through the use of gene editing tools we will see a dramatic increase in animal model resources available for both the study of human disease and the translation of this knowledge into the clinic. Comparative pathology will be central to the productive use of these animal models and the successful translation of new therapeutic strategies.

Potential of gene drives with genome editing to increase genetic gain in livestock breeding programs.

BACKGROUND: This paper uses simulation to explore how gene drives can increase genetic gain in livestock breeding programs. Gene drives are naturally occurring phenomena that cause a mutation on one chromosome to copy itself onto its homologous chromosome. METHODS: We simulated nine different breeding and editing scenarios with a common overall structure. Each scenario began with 21 generations of selection, followed by 20 generations of selection based on true breeding values where the breeder used selection alone, selection in combination with genome editing, or selection with genome editing and gene drives. In the scenarios that used gene drives, we varied the probability of successfully incorporating the gene drive. For each scenario, we evaluated genetic gain, genetic variance [Formula: see text], rate of change in inbreeding ([Formula: see text]), number of distinct quantitative trait nucleotides (QTN) edited, rate of increase in favourable allele frequencies of edited QTN and the time to fix favourable alleles. RESULTS: Gene drives enhanced the benefits of genome editing in seven ways: (1) they amplified the increase in genetic gain brought about by genome editing; (2) they amplified the rate of increase in the frequency of favourable alleles and reduced the time it took to fix them; (3) they enabled more rapid targeting of QTN with lesser effect for genome editing; (4) they distributed fixed editing resources across a larger number of distinct QTN across generations; (5) they focussed editing on a smaller number of QTN within a given generation; (6) they reduced the level of inbreeding when editing a subset of the sires; and (7) they increased the efficiency of converting genetic variation into genetic gain. CONCLUSIONS: Genome editing in livestock breeding results in short-, medium- and long-term increases in genetic gain. The increase in genetic gain occurs because editing increases the frequency of favourable alleles in the population. Gene drives accelerate the increase in allele frequency caused by editing, which results in even higher genetic gain over a shorter period of time with no impact on inbreeding.

Gene targeting, genome editing: from Dolly to editors.

One of the most powerful strategies to investigate biology we have as scientists, is the ability to transfer genetic material in a controlled and deliberate manner between organisms. When applied to livestock, applications worthy of commercial venture can be devised. Although initial methods used to generate transgenic livestock resulted in random transgene insertion, the development of SCNT technology enabled homologous recombination gene targeting strategies to be used in livestock. Much has been accomplished using this approach. However, now we have the ability to change a specific base in the genome without leaving any other DNA mark, with no need for a transgene. With the advent of the genome editors this is now possible and like other significant technological leaps, the result is an even greater diversity of possible applications. Indeed, in merely 5 years, these 'molecular scissors' have enabled the production of more than 300 differently edited pigs, cattle, sheep and goats. The advent of genome editors has brought genetic engineering of livestock to a position where industry, the public and politicians are all eager to see real use of genetically engineered livestock to address societal needs. Since the first transgenic livestock reported just over three decades ago the field of livestock biotechnology has come a long way-but the most exciting period is just starting.

Genetically engineering milk.

It has been thirty years since the first genetically engineered animal with altered milk composition was reported. During the intervening years, the world population has increased from 5bn to 7bn people. An increasing demand for protein in the human diet has followed this population expansion, putting huge stress on the food supply chain. Many solutions to the grand challenge of food security for all have been proposed and are currently under investigation and study. Amongst these, genetics still has an important role to play, aiming to continually enable the selection of livestock with enhanced traits. Part of the geneticist's tool box is the technology of genetic engineering. In this Invited Review, we indicate that this technology has come a long way, we focus on the genetic engineering of dairy animals and we argue that the new strategies for precision breeding demand proper evaluation as to how they could contribute to the essential increases in agricultural productivity our society must achieve.

Genome edited sheep and cattle.

Genome editing tools enable efficient and accurate genome manipulation. An enhanced ability to modify the genomes of livestock species could be utilized to improve disease resistance, productivity or breeding capability as well as the generation of new biomedical models. To date, with respect to the direct injection of genome editor mRNA into livestock zygotes, this technology has been limited to the generation of pigs with edited genomes. To capture the far-reaching applications of gene-editing, from disease modelling to agricultural improvement, the technology must be easily applied to a number of species using a variety of approaches. In this study, we demonstrate zygote injection of TALEN mRNA can also produce gene-edited cattle and sheep. In both species we have targeted the myostatin (MSTN) gene. In addition, we report a critical innovation for application of gene-editing to the cattle industry whereby gene-edited calves can be produced with specified genetics by ovum pickup, in vitro fertilization and zygote microinjection (OPU-IVF-ZM). This provides a practical alternative to somatic cell nuclear transfer for gene knockout or introgression of desirable alleles into a target breed/genetic line.

Highly efficient targeted chromosome deletions using CRISPR/Cas9.

The CRISPR/Cas9 system has emerged as an intriguing new technology for genome engineering. It utilizes the bacterial endonuclease Cas9 which, when delivered to eukaryotic cells in conjunction with a user-specified small guide RNA (gRNA), cleaves the chromosomal DNA at the target site. Here we show that concurrent delivery of gRNAs designed to target two different sites in a human chromosome introduce DNA double-strand breaks in the chromosome and give rise to targeted deletions of the intervening genomic segment. Predetermined genomic DNA segments ranging from several-hundred base pairs to 1 Mbp can be precisely deleted at frequencies of 1-10%, with no apparent correlation between the size of the deleted fragment and the deletion frequency. The high efficiency of this technique holds promise for large genomic deletions that could be useful in generation of cell and animal models with engineered chromosomes.

Potential of promotion of alleles by genome editing to improve quantitative traits in livestock breeding programs.

BACKGROUND: Genome editing (GE) is a method that enables specific nucleotides in the genome of an individual to be changed. To date, use of GE in livestock has focussed on simple traits that are controlled by a few quantitative trait nucleotides (QTN) with large effects. The aim of this study was to evaluate the potential of GE to improve quantitative traits that are controlled by many QTN, referred to here as promotion of alleles by genome editing (PAGE). METHODS: Multiple scenarios were simulated to test alternative PAGE strategies for a quantitative trait. They differed in (i) the number of edits per sire (0 to 100), (ii) the number of edits per generation (0 to 500), and (iii) the extent of use of PAGE (i.e. editing all sires or only a proportion of them). The base line scenario involved selecting individuals on true breeding values (i.e., genomic selection only (GS only)-genomic selection with perfect accuracy) for several generations. Alternative scenarios complemented this base line scenario with PAGE (GS + PAGE). The effect of different PAGE strategies was quantified by comparing response to selection, changes in allele frequencies, the number of distinct QTN edited, the sum of absolute effects of the edited QTN per generation, and inbreeding. RESULTS: Response to selection after 20 generations was between 1.08 and 4.12 times higher with GS + PAGE than with GS only. Increases in response to selection were larger with more edits per sire and more sires edited. When the total resources for PAGE were limited, editing a few sires for many QTN resulted in greater response to selection and inbreeding compared to editing many sires for a few QTN. Between the scenarios GS only and GS + PAGE, there was little difference in the average change in QTN allele frequencies, but there was a major difference for the QTN with the largest effects. The sum of the effects of the edited QTN decreased across generations. CONCLUSIONS: This study showed that PAGE has great potential for application in livestock breeding programs, but inbreeding needs to be managed.

Efficient TALEN-mediated gene knockout in livestock.

Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Coselection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

Site specific insertion of a transgene into the murine α-casein (CSN1S1) gene results in the predictable expression of a recombinant protein in milk.

Gene loci of highly expressed genes provide ideal sites for transgene expression. Casein genes are highly expressed in mammals leading to the synthesis of substantial amounts of casein proteins in milk. The α-casein (CSN1S1) gene has assessed as a site of transgene expression in transgenic mice and a mammary gland cell line. A transgene encoding an antibody light chain gene (A1L) was inserted into the α-casein gene using sequential homologous and site-specific recombination. Expression of the inserted transgene is directed by the α-casein promoter, is responsive to lactogenic hormone activation, leads to the synthesis of a chimeric α-casein/A1L transgene mRNA, and secretion of the recombinant A1L protein into milk. Transgene expression is highly consistent in all transgenic lines, but lower than that of the α-casein gene (4%). Recombinant A1L protein accounted for 0.5% and 1.6% of total milk protein in heterozygous and homozygous transgenic mice, respectively. The absence of the α-casein protein in homozygous A1L transgenic mice leads to a reduction of total milk protein and delayed growth of the pups nursed by these mice. Overall, the data demonstrate that the insertion of a transgene into a highly expressed endogenous gene is insufficient to guarantee its abundant expression.

TT2013 meeting report: the Transgenic Technology meeting visits Asia for the first time.

The 11th Transgenic Technology meeting was held in Guangzhou, China on 25th-27th February 2013. Over 300 scientists and students from round the world gathered to hear the latest developments in the technologies underpinning the creation of transgenic and knockout animals and their application to biological sciences in areas such as the modeling human diseases and biotechnology. As well as informative presentations from leading researchers in the field, an excellent selection of short talks selected from abstracts and posters, attendees were also treated to an inspiring talk from Allan Bradley who was awarded the 9th International Society of Transgenic Technologies Prize for outstanding contributions to the field of transgenic technologies.

Novel GM animal technologies and their governance.

Scientific advances in methods of producing genetically modified (GM) animals continue, yet few such animals have reached commercial production. Existing regulations designed for early techniques of genetic modification pose formidable barriers to commercial applications. Radically improved techniques for producing GM animals invite a re-examination of current regulatory regimes. We critically examine current GM animal regulations, with a particular focus on the European Union, through a framework that recognises the importance of interactions among regulatory regimes, innovation outcomes and industry sectors. The current focus on the regulation of risk is necessary but is unable to discriminate among applications and tends to close down broad areas of application rather than facilitate innovation and positive industry interactions. Furthermore, the fields of innovative animal biosciences appear to lack networks of organisations with co-ordinated future oriented actions. Such networks could drive coherent programmes of innovation towards particular visions and contribute actively to the development of regulatory systems for GM animals. The analysis presented makes the case for regulatory consideration of each animal bioscience related innovation on the basis of the nature of the product itself and not the process by which it was developed.

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice.

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn't fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.

Mammary gland development is delayed in mice deficient for aminopeptidase N.

Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.

Species-specific variation in RELA underlies differences in NF-κB activity: a potential role in African swine fever pathogenesis.

African swine fever virus (ASFV) is a highly infectious disease of domestic pigs, with virulent isolates causing a rapidly fatal hemorrhagic fever. In contrast, the porcine species endogenous to Africa tolerate infection. The ability of the virus to persist in one host while killing another genetically related host implies that disease severity may be, in part, modulated by host genetic variation. To complement transcription profiling approaches to identify the underlying genetic variation in the host response to ASFV, we have taken a candidate gene approach based on known signaling pathways that interact with the virus-encoded immunomodulatory protein A238L. We report the sequencing of these genes from different pig species and the identification and initial in vitro characterization of polymorphic variation in RELA (p65; v-rel reticuloendotheliosis viral oncogene homolog A), the major component of the NF-κB transcription factor. Warthog RELA and domestic pig RELA differ at three amino acids. Transient cell transfection assays indicate that this variation is reflected in reduced NF-κB activity in vitro for warthog RELA but not for domestic pig RELA. Induction assays indicate that warthog RELA and domestic pig RELA are elevated essentially to the same extent. Finally, mutational studies indicate that the S531P site conveys the majority of the functional variation between warthog RELA and domestic pig RELA. We propose that the variation in RELA identified between the warthog and domestic pig has the potential to underlie the difference between tolerance and rapid death upon ASFV infection.