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.Tal-effector nuclease | biotechnology | gene-editing

Sheep are a key source of meat, milk and fibre for the global livestock sector, and an important biomedical model. Global analysis of gene expression across multiple tissues has aided genome annotation and supported functional annotation of mammalian genes. We present a large-scale RNA-Seq dataset representing all the major organ systems from adult sheep and from several juvenile, neonatal and prenatal developmental time points. The Ovis aries reference genome (Oar v3.1) includes 27,504 genes (20,921 protein coding), of which 25,350 (19,921 protein coding) had detectable expression in at least one tissue in the sheep gene expression atlas dataset. Network-based cluster analysis of this dataset grouped genes according to their expression pattern. The principle of ‘guilt by association’ was used to infer the function of uncharacterised genes from their co-expression with genes of known function. We describe the overall transcriptional signatures present in the sheep gene expression atlas and assign those signatures, where possible, to specific cell populations or pathways. The findings are related to innate immunity by focusing on clusters with an immune signature, and to the advantages of cross-breeding by examining the patterns of genes exhibiting the greatest expression differences between purebred and crossbred animals. This high-resolution gene expression atlas for sheep is, to our knowledge, the largest transcriptomic dataset from any livestock species to date. It provides a resource to improve the annotation of the current reference genome for sheep, presenting a model transcriptome for ruminants and insight into gene, cell and tissue function at multiple developmental stages.

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.

Mutations in C9ORF72 are the most common cause of familial amyotrophic lateral sclerosis (ALS). Here, through a combination of RNA-Seq and electrophysiological studies on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs), we show that increased expression of GluA1 AMPA receptor (AMPAR) subunit occurs in MNs with C9ORF72 mutations that leads to increased Ca2+-permeable AMPAR expression and results in enhanced selective MN vulnerability to excitotoxicity. These deficits are not found in iPSC-derived cortical neurons and are abolished by CRISPR/Cas9-mediated correction of the C9ORF72 repeat expansion in MNs. We also demonstrate that MN-specific dysregulation of AMPAR expression is also present in C9ORF72 patient post-mortem material. We therefore present multiple lines of evidence for the specific upregulation of GluA1 subunits in human mutant C9ORF72 MNs that could lead to a potential pathogenic excitotoxic mechanism in ALS.

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.

BackgroundGenome 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).MethodsMultiple 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.ResultsResponse 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.ConclusionsThis study showed that PAGE has great potential for application in livestock breeding programs, but inbreeding needs to be managed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12711-015-0135-3) contains supplementary material, which is available to authorized users.

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.

Transcription of tissue-specific genes in mammary gland requires signals from both prolactin and basement membrane. Here we address the mechanism by which this specialized extracellular matrix regulates transcription. Using mammary cell cultures derived from transgenic mice harboring the ovine ␤-lactoglobulin gene, we show that either a basement membrane extract, or purified laminin-1, induced high levels of ␤-lactoglobulin synthesis. It is known that prolactin signals through Stat5 (signal transducer and activator of transcription). This transcription factor interacts with ␥-interferon activation site-related motifs within the ␤-lactoglobulin promoter, which we show are required for matrix dependence of ␤-lactoglobulin expression. The DNA binding activity of Stat5 was present only in extracts of mammary cells cultured on basement membrane, indicating that the activation state of Stat5 is regulated by the type of substratum the cell encounters. Thus, basement membrane controls transcription of milk protein genes through the Stat5-mediated prolactin signaling pathway, providing a molecular explanation for previous studies implicating extracellular matrix in the control of mammary differentiation.Cell-matrix interactions are critical for regulating the phenotype of many cells. In mammary gland, basement membrane is necessary for the prolactin-mediated control of lactation (1-3). However, the mechanism by which extracellular matrix (ECM) 1 influences differentiation in mammary epithelial cells has not been elucidated. We have shown previously that functional ␤ 1 integrins are required for mammary differentiation (4) and that the basement membrane component laminin-1 directs milk protein gene transcription coordinately with prolactin (5).The prolactin pathway is driven through the protein tyrosine kinase Jak2 (6 -10) and one of its substrates, the transcription factor Stat5 (11,12). Stat factors associate with cytokine receptors following ligand binding and subsequently become phosphorylated by receptor-associated Jaks. They then dimerize and translocate to the nucleus where they bind specific DNA sequence motifs, thus contributing to transcriptional activation (13)(14)(15).In this paper we examine whether an element of the prolactin signaling pathway is modulated directly by cell-matrix interactions, thereby mediating the ECM control of transcription. Using primary and first passage cultures of mammary epithelial cells, we demonstrate that the activity of the promoter for the milk protein ␤-lactoglobulin (BLG) is dependent on basement membrane and that Stat5 recognition sites within this promoter are required for transcription. Moreover, we show that the ability of Stat5 to bind its cognate DNA sequence within the BLG promoter requires cell interactions with both basement membrane and prolactin. Thus, matrix and cytokine signals converge on a single pathway.Our data demonstrate, for the first time, that the activity of a Stat transcription factor is a target for regulation by ECM. This establishes a novel signaling...