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Indirect modification of animal genomes by interspecific hybridization, cross-breeding, and selection has produced an enormous spectrum of phenotypic diversity over more than 10,000 yr of animal domestication. Using these established technologies, the farming community has successfully increased the yield and efficiency of production in most agricultural species while utilizing land resources that are often unsuitable for other agricultural purposes. Moving forward, animal well-being and agricultural sustainability are moral and economic priorities of consumers and producers alike. Therefore, these considerations will be included in any strategy designed to meet the challenges produced by global climate change and an expanding world population. Improvements in the efficiency and precision of genetic technologies will enable a timely response to meet the multifaceted food requirements of a rapidly increasing world population.

The zinc finger transcription factor PRDM14, part of the PR domain containing protein family, is critical for mammalian primordial germ cell (PGC) specification, epigenetic reprogramming and maintaining naïve pluripotency in stem cells. However, PRDM14's role in other species is not well understood. In chicken, PRDM14 is broadly expressed in the early embryo, before becoming restricted to the forming neural plate, migratory PGCs, and later, in the adult testes. To investigate the role of PRDM14 we generated two independent targeted chicken lines and bred homozygous knockout embryos. Strikingly, we found that gastrulation was disrupted in PRDM14 -/- embryos, which lacked a definitive primitive streak. Transcriptomic and in situ hybridisation analyses revealed a broad loss of anterior primitive streak marker genes, coupled with downregulation of the multifunctional antagonists CHRD and CER1, and expansion of the NODAL expression domain. Further analysis of PRDM14 -/- embryos revealed PGCs were still specified but significantly reduced in number, and PRDM14 -/- PGCs could not be propagated in vitro. Knockdown studies in vitro confirmed that PRDM14 is essential for PGC survival and antagonises FGF-induced somatic differentiation, similar to PRDM14's role in mammalian stem cells. Taken together, our results show that in chicken, PRDM14 plays a multifunctional and essential role during embryonic development., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

Following infection of a porcine dam with PRRSV around 90 days of gestation, the virus crosses the placenta and starts to infect fetuses. This can lead to consequences such as abortions, stillbirths, and respiratory issues in newborn piglets. CD163 is an essential cellular viral entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Gene-edited pigs possessing a complete deletion of CD163 are resistant to PRRSV infection. Recently, we demonstrated that pigs harboring a clean deletion of CD163 exon 13 (ΔExon13 CD163 pigs) which encodes the first 12 amino acids of the CD163 PSTII domain were not susceptible to PRRSV infection. In this study, ΔExon13 CD163 (-/-) gilts were bred with wildtype CD163 (+/+) boars producing heterozygous, CD163 (+/-) fetuses. We found that fetuses with a wildtype CD163, recovered between day 103 of gestation or 17 days after the maternal infection with PRRSV, were fully protected from PRRSV in dams containing a clean deletion of CD163 exon 13. These findings suggest a feasible approach for eliminating PRRSV-related reproductive illness, which is a significant cause of economic losses in agriculture., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The work is in support of an ongoing U.S. patent application., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Global warming is a major challenge to the sustainable and humane production of food because of the increased risk of livestock to heat stress. Here, the example of the prolactin receptor ( PRLR ) gene is used to demonstrate how gene editing can increase the resistance of cattle to heat stress by the introduction of mutations conferring thermotolerance. Several cattle populations in South and Central America possess natural mutations in PRLR that result in affected animals having short hair and being thermotolerant. CRISPR/Cas9 technology was used to introduce variants of PRLR in two thermosensitive breeds of cattle - Angus and Jersey. Gene-edited animals exhibited superior ability to regulate vaginal temperature (heifers) and rectal temperature (bulls) compared to animals that were not gene-edited. Moreover, gene-edited animals exhibited superior growth characteristics and had larger scrotal circumference. There was no evidence for deleterious effects of the mutation on carcass characteristics or male reproductive function. These results indicate the potential for reducing heat stress in relevant environments to enhance cattle productivity., Competing Interests: Authors from Acceligen and Semex have an interest in commercialization of gene‐edited livestock. There are no other conflicts., (©2024 The Authors FASEB BioAdvances published by The Federation of American Societies for Experimental Biology.)

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The work is in support of an ongoing U.S. patent application., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Spermatogonial stem cell (SSC) transplantation into the testis of a germ cell (GC)-depleted surrogate allows transmission of donor genotype via donor-derived sperm produced by the recipient. Transplantation of gene-edited SSCs provides an approach to propagate gene-edited large animal models. DAZL is a conserved RNA-binding protein important for GC development, and DAZL knockout (KO) causes defects in GC commitment and differentiation. We characterized DAZL -KO pigs as SSC transplantation recipients. While there were GCs in 1-week-old (wko) KO, complete GC depletion was observed by 10 wko. Donor GCs were transplanted into 18 DAZL -KO recipients at 10-13 wko. At sexual maturity, semen and testes were evaluated for transplantation efficiency and spermatogenesis. Approximately 22% of recipient seminiferous tubules contained GCs, including elongated spermatids and proliferating spermatogonia. The ejaculate of 89% of recipients contained sperm, exclusively from donor origin. However, sperm concentration was lower than the wild-type range. Testicular protein expression and serum hormonal levels were comparable between DAZL -KO and wild-type. Intratesticular testosterone and Leydig cell volume were increased, and Leydig cell number decreased in transplanted DAZL -KO testis compared to wild-type. In summary, DAZL -KO pigs support donor-derived spermatogenesis following SSC transplantation, but low spermatogenic efficiency currently limits their use for the production of offspring.

Research, monitoring, and management of marine and aquatic ecosystems often require surface water samples to measure biogeochemical and optical parameters. Traditional sampling with a boat and several personnel onboard can be labor-intensive and safety requirements limit sampling activities in high-risk environments. This paper describes the Naval Operating Research Drone Assessing Climate Change (NORDACC). NORDACC is an open source, light-weight, and portable autonomous surface vehicle that can acquire surface water samples while also measuring sea surface temperature and salinity for the duration of its deployment. NORDACC is ideal for operations in remote areas where resources and personnel are limited. Two sample bottles, each one liter in volume, can be filled, either at pre-programmed sampling stations or manually, using the remote control. A trimaran design provides buoyancy and stability, with hulls constructed of vacuum-formed acrylonitrile butadiene styrene (ABS) plastic. NORDACC can navigate autonomously between waypoints and features first person view capabilities for enhanced situational awareness. NORDACC's performance was validated in Aarhus Bay, Denmark, collecting multiple surface water samples in winds in excess of 8 ms -1 and steep, choppy waves., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Despite growing attention on the contribution of macroalgae to carbon cycling and sequestration (blue carbon), more observational data is needed to constrain current estimates. In this study, we estimate the floating macroalgal carbon flux within and beyond a large sub-Arctic fjord system, Nuup Kangerlua, Greenland, which could potentially reach carbon sinks. Our study estimates 1) the fjord-scale area with macroalgal coverage and barrens caused by sea urchin grazing, 2) the floating macroalgal biomass in the fjord, and 3) the annual export flux of floating macroalgae out of the fjord system. ROV surveys documented that macroalgal habitats cover 32 % of the seafloor within the photic zone (0-30 m) with an average coverage of 39.6, 22, and 7.2 % in the depth intervals 0-10, 10-20, and 20-30 m, respectively. 15 % of the area suitable for macroalgae was denuded by sea urchin grazing. Floating macroalgae were common with an average biomass of 55 kg wet weight km -2 . Densities and species composition varied seasonally with the highest levels after storms. The floating biomass was composed of intertidal macroalgal species (58 %) (Fucus vesiculosus, Fucus distichus, and Ascophyllum nodosum) and kelps (42 %) (Saccharina longicruris, S. latissima, and Alaria esculenta). We deployed surface GPS drifters to simulate floating macroalgal trajectories and velocity. Data indicated that 80 % of the floating biomass is retained in the fjord where its fate in relation to long-term sequestration is unknown. Export beyond the fjord was limited and indicated an annual floating macroalgal export beyond the fjord of only 6.92 t C yr -1 , which is equal to ~0.02 % of the annual net primary production. Our findings suggest that floating macroalgae support a limited blue carbon potential beyond this fjord and that future research should focus on the fate of retained floating macroalgae and subsurface export to resolve the connectivity between macroalgal habitats and long-term carbon sinks., Competing Interests: Declaration of competing interest We have no competing interests., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones., (Published by Oxford University Press on behalf of National Academy of Sciences 2023.)

Changes in the distribution of coastal macrophytes in Greenland, and elsewhere in the Arctic are difficult to quantify as the region remains challenging to access and monitor. Satellite imagery, in particular Sentinel-2 (S2), may enable large-scale monitoring of coastal areas in Greenland but its use is impacted by the optically complex environments and the scarcity of supporting data in the region. Additionally, the canopies of the dominant macrophyte species in Greenland do not extend to the sea surface, limiting the use of indices that exploit the reflection of near-infrared radiation by vegetation due to its absorption by seawater. Three hypotheses are tested: I) 10-m S2 imagery and commonly used detection methods can identify intertidal macrophytes that are exposed at low tide in an optically complex fjord system in Greenland impacted by marine and land terminating glaciers; II) detached and floating macrophytes accumulate in patches that are sufficiently large to be detected by 10-m S2 images; III) iceberg scour and/or turbid meltwater runoff shape the spatial distribution of intertidal macroalgae in fjord systems with marine-terminating glaciers. The NDVI produced the best results in optically complex fjord systems in Greenland. 12 km 2 of exposed intertidal macrophytes were identified in the study area at low tide. Floating mats of macrophytes ranged in area from 400 m 2 to 326,800 m 2 and were most common at the mouth of the fjord. Icebergs and turbidity appear to play a role in structuring the distribution of intertidal macrophytes and the retreat of marine terminating glaciers could allow macrophytes cover to expand. The challenges and solutions presented here apply to most fjords in Greenland and, therefore, the methodology may be extended to produce a Greenland-wide estimate of intertidal macrophytes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration. Methods: To test this hypothesis, we generated the first KISS1 -edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed. Results: Thirty-five of these pigs bore KISS1 -disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1- edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1 . Founder pigs partially carrying KISS1 -disruptive alleles were bred resulting in a total of 53 KISS1 +/+ , 60 KISS1 +/- , and 34 KISS1 -/- F1 liveborn piglets, confirming germline transmission. Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration., Competing Interests: The authors JF, KM, JB, PR-V, FO, SL, E-SK, and TS are employed by Acceligen Inc., a wholly owned subsidiary of Recombinetics Inc.; SS, UG, AC, and DC are employed by Recombinetics Inc. AH is an employee of Hendrix Genetics. These companies contributed supplemental funding for this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Flórez, Martins, Solin, Bostrom, Rodríguez-Villamil, Ongaratto, Larson, Ganbaatar, Coutts, Kern, Murphy, Kim, Carlson, Huisman, Sonstegard and Lents.)

As the genetic mutations driving human disease are identified, there is an increasing need for a biomedical model that can accurately represent the disease of interest and provide a platform for potential therapeutic testing. Pigs are a better model for human disease than rodents because of their genetic and physiological similarities to humans. However, current methods to generate porcine models are both technically challenging and expensive. Germline genetic modification through gene edited spermatogonia provides an effective alternative to how these models are developed. Here, we report an improved technique of gene editing in spermatogonia of pigs using CRISPR-Cas9 to generate different edits that reflect the genotypes of human diseases., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Bovine viral diarrhea virus's (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12 , GRID2 , and RABGAP1L . However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12 , GRID2 , or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Background: There are over 17,000 patients in the US waiting to receive liver transplants, and these numbers are increasing dramatically. Significant effort is being made to obtain functional hepatocytes and liver tissue that can for therapeutic use in patients. Blastocyst complementation is a challenging, innovative technology that could fundamentally change the future of organ transplantation. It requires the knockout (KO) of genes essential for cell or organ development in early stage host embryos followed by injection of donor pluripotent stem cells (PSCs) into host blastocysts to generate chimeric offspring in which progeny of the donor cells populate the open niche to develop functional tissues and organs., Methods: The HHEX gene is necessary for proper liver development. We engineered loss of HHEX gene expression in early mouse and pig embryos and performed intraspecies blastocyst complementation of HHEX KO embryos with eGFP-labeled PSCs in order to rescue the loss of liver development., Results: Loss of HHEX gene expression resulted in embryonic lethality at day 10.5 in mice and produced characteristics of lethality at day 18 in pigs, with absence of liver tissue in both species. Analyses of mouse and pig HHEX KO fetuses confirmed significant loss of liver-specific gene and protein expression. Intraspecies blastocyst complementation restored liver formation and liver-specific proteins in both mouse and pig. Livers in complemented chimeric fetuses in both species were comprised of eGFP-labeled donor-derived cells and survived beyond the previously observed time of HHEX KO embryonic lethality., Conclusions: This work demonstrates that loss of liver development in the HHEX KO can be rescued via blastocyst complementation in both mice and pigs. This complementation strategy is the first step towards generating interspecies chimeras for the goal of producing human liver cells, tissues, and potentially complete organs for clinical transplantation.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

To study the pathophysiology of human diseases, develop innovative treatments, and refine approaches for regenerative medicine require appropriate preclinical models. Pigs share physiologic and anatomic characteristics with humans and are genetically more similar to humans than are mice. Genetically modified pigs are essential where rodent models do not mimic the human disease phenotype. The male germline stem cell or spermatogonial stem cell (SSC) is unique; it is the only cell type in an adult male that divides and contributes genes to future generations, making it an ideal target for genetic modification. Here we report that CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing in porcine spermatogonia that include SSCs is significantly more efficient than previously reported editing with TALENs and allows precise gene editing by homology directed repair (HDR). We also established homology-mediated end joining (HMEJ) as a second approach to targeted gene editing to enable introduction of larger transgenes and/or humanizing parts of the pig genome for disease modeling or regenerative medicine. In summary, the approaches established in the current study result in efficient targeted genome editing in porcine germ cells for precise replication of human disease alleles., Competing Interests: DW, SS, TG, DC, and ID are employees and/or shareholders in Recombinetics, Inc., a company that commercializes gene editing in livestock. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Webster, Bondareva, Solin, Goldsmith, Su, Lara, Carlson and Dobrinski.)

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele., Competing Interests: DRK, DFC, ALW, and DAW are employed by Recombinetics, Inc., which controls the commercial rights to the animal model. The conflict had no impact on this study and does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

Background & Aims: Microvillus inclusion disease (MVID) is caused by inactivating mutations in the myosin VB gene (MYO5B). MVID is a complex disorder characterized by chronic, watery, life-threatening diarrhea that usually begins in the first hours to days of life. We developed a large animal model of MVID to better understand its pathophysiology., Methods: Pigs were cloned by transfer of chromatin from swine primary fetal fibroblasts, which were edited with TALENs and single-strand oligonucleotide to introduce a P663-L663 substitution in the endogenous swine MYO5B (corresponding to the P660L mutation in human MYO5B, associated with MVID) to fertilized oocytes. We analyzed duodenal tissues from patients with MVID (with the MYO5B P660L mutation) and without (controls), and from pigs using immunohistochemistry. Enteroids were generated from pigs with MYO5B(P663L) and without the substitution (control pigs)., Results: Duodenal tissues from patients with MVID lacked MYO5B at the base of the apical membrane of intestinal cells; instead MYO5B was intracellular. Intestinal tissues and derived enteroids from MYO5B(P663L) piglets had reduced apical levels and diffuse subapical levels of sodium hydrogen exchanger 3 and SGLT1, which regulate transport of sodium, glucose, and water, compared with tissues from control piglets. However, intestinal tissues and derived enteroids from MYO5B(P663L) piglets maintained CFTR on apical membranes, like tissues from control pigs. Liver tissues from MYO5B(P663L) piglets had alterations in bile salt export pump, a transporter that facilitates bile flow, which is normally expressed in the bile canaliculi in the liver., Conclusions: We developed a large animal model of MVID that has many features of the human disease. Studies of this model could provide information about the functions of MYO5B and MVID pathogenesis, and might lead to new treatments., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems., Competing Interests: WW Interests in Lifengine and Lifengine Animal Health, JW, MA, CM, MT, TW, SK, MV, ML, KM, JL, ZM, AW, CM, JH, KK, CC, DB, BW, BM, DD, MM No competing interests declared, DW, SS, DC Shares in Recombinetics, Inc, SE Shares in Lifengine, and Lifengine Animal Health, KC Shares in Recombinetics, Inc, Lifengine and Lifengine Animal Health, JE JJE has a financial conflict of interest with Recombinetics, Inc; Immusoft, Inc; LifEngine and LifEngine Animal Technologies;, (© 2020, Wierson et al.)

Most mammalian cells possess a single, non-motile primary cilium that plays an important role in mediating cellular signaling pathways, such as Hedgehog (Hh) signaling. Primary cilia are present on testicular somatic cells and demonstrate a temporal expression during development; however, their role in testicular morphogenesis is not well characterized. To investigate the role of primary cilia and Hh signaling in Sertoli cells on morphogenesis, we inhibited assembly of primary cilia through CRISPR Cas9-mediated gene editing of ODF2, a structural component of primary cilia and siRNA-mediated gene silencing of IFT88, a functional component of the intraflagellar transport system. Knockdown of ODF2 and IFT88 resulted in a 50% reduction in the number of cells with primary cilia and significant shortening of the remaining cilia. The expression of GLI1, a downstream target of Hh signaling, was significantly reduced when IFT88 but not ODF2, was downregulated. When morphogenesis was examined using tubule formation in vitro and a novel testicular organoid system, loss of cilia after knockdown of both targets affected cellular assembly and organization. While the Hh pathway was found to be active during morphogenesis in vitro, addition of the Hh antagonist cyclopamine did not affect morphogenesis in either in vitro system. These results indicate that primary cilia are important for morphogenesis in vitro but Hh signaling is not the cilia-mediated pathway responsible for orchestrating morphogenic organization.

Background: Heart rate variability (HRV) and pulse rate variability are indices of autonomic cardiac modulation. Increased pericardial fat is associated with worse cardiovascular outcomes. We hypothesized that progressive increases in pericardial fat volume and inflammation prospectively dampen HRV in hypercholesterolemic pigs., Methods: WT (wild type) or PCSK9 (proprotein convertase subtilisin-like/kexin type-9) gain-of-function Ossabaw mini-pigs were studied in vivo before and after 3 and 6 months of a normal diet (WT-normal diet, n=4; PCSK9-normal diet, n=6) or high-fat diet (HFD; WT-HFD, n=3; PCSK9-HFD, n=6). The arterial pulse waveform was obtained from an arterial telemetry transmitter to analyze HRV indices, including SD (SD of all pulse-to-pulse intervals over a single 5-minute period), root mean square of successive differences, proportion >50 ms of normal-to-normal R-R intervals, and the calculated ratio of low-to-high frequency distributions (low-frequency power/high-frequency power). Pericardial fat volumes were evaluated using multidetector computed tomography and its inflammation by gene expression of TNF (tumor necrosis factor)-α. Plasma lipid panel and norepinephrine level were also measured., Results: At diet completion, hypercholesterolemic PCSK9-HFD had significantly ( P <0.05 versus baseline) depressed HRV (SD of all pulse-to-pulse intervals over a single 5-minute period, root mean square of successive differences, proportion >50 ms, high-frequency power, low-frequency power), and both HFD groups had higher sympathovagal balance (SD of all pulse-to-pulse intervals over a single 5-minute period/root mean square of successive differences, low-frequency power/high-frequency power) compared with normal diet. Pericardial fat volumes and LDL (low-density lipoprotein) cholesterol concentrations correlated inversely with HRV and directly with sympathovagal balance, while sympathovagal balance correlated directly with plasma norepinephrine. Pericardial fat TNF-α expression was upregulated in PCSK9-HFD, colocalized with nerve fibers, and correlated inversely with root mean square of successive differences and proportion >50 ms., Conclusions: Progressive pericardial fat expansion and inflammation are associated with a fall in HRV in Ossabaw mini-pigs, implying aggravated autonomic imbalance. Hence, pericardial fat accumulation is associated with alterations in HRV and the autonomic nervous system. Visual Overview: A visual overview is available for this article.

Icebergs account for approximately half of the freshwater flux from the Greenland Ice Sheet and they can impact marine ecosystems by releasing nutrients and sediments into the ocean as they drift and melt. Parameterizing iceberg fluxes of nutrients and sediments to fjord and ocean waters remains a difficult task due to the complexity of ice-ocean interactions and is complicated by a lack of observations. Acquiring iceberg samples can be difficult and dangerous, as icebergs can break apart and roll without warning. Here we present open source design files for a small, lightweight ice coring drill that can be reproduced using modern computer numerical control (CNC) machining and 3D printing technology. This ice core drill can rapidly acquire small ice samples from icebergs and bergy bits using a standard commercial, off-the-shelf battery-operated hand drill. Design files and a recent field expedition to Northwest Greenland are described. Ice core collection required only 30 s, thereby minimizing risks to scientists., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Meltwater runoff from the Greenland Ice Sheet changes water levels in glacial lakes and can lead to glacial lake outburst flooding (GLOF) events that threaten lives and property. Icebergs produced at Greenland's marine terminating glaciers drift into Baffin Bay and the North Atlantic, where they can threaten shipping and offshore installations. Thus, monitoring glacial lake water levels and the drift of icebergs can enhance safety and aid in the scientific studies of glacial hydrology and iceberg-ocean interactions. The Maker Buoy was originally designed as a low-cost and open source sensor to monitor surface ocean currents. The open source framework, low-cost components, rugged construction and affordable satellite data transmission capabilities make it easy to customize for environmental monitoring in remote areas and under harsh conditions. Here, we present two such Maker Buoy variants that were developed to monitor water level in an ice-infested glacial lake in southern Greenland and to track drifting icebergs and moorings in the Vaigat Strait (Northwest Greenland). We describe the construction of each design variant, methods to access data in the field without an internet connection, and deployments in Greenland in summer 2019. The successful deployments of each Maker Buoy variant suggest that they may also be useful in operational iceberg management strategies and in GLOF monitoring programs.

Rat embryonic stem cells (rESCs) are capable of contributing to all differentiated tissues, including the germ line in chimeric animals, and represent a unique, authentic alternative to mouse embryonic stem cells for studying stem cell pluripotency and self-renewal. Here, we describe an EGFP reporter transgene that tracks expression of the benchmark naive pluripotency marker gene Rex1 (Zfp42) in the rat. Insertion of the EGFP reporter gene downstream of the Rex1 promoter disrupted Rex1 expression, but REX1-deficient rESCs and rats were viable and apparently normal, validating this targeted knockin transgene as a neutral reporter. The Rex1-EGFP gene responded to self-renewal/differentiation factors and validated the critical role of β-catenin/LEF1 signaling. The stem cell reporter also allowed the identification of functionally distinct sub-populations of cells within rESC cultures, thus demonstrating its utility in discriminating between cell states in rat stem cell cultures, as well as providing a tool for tracking Rex1 expression in the rat., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mammalian primordial germ cells (PGCs) are induced in the embryonic epiblast, before migrating to the nascent gonads. In fish, frogs, and birds, the germline segregates even earlier, through the action of maternally inherited germ plasm. Across vertebrates, migrating PGCs retain a broad developmental potential, regardless of whether they were induced or maternally segregated. In mammals, this potential is indicated by expression of pluripotency factors, and the ability to generate teratomas and pluripotent cell lines. How the germline loses this developmental potential remains unknown. Our genome-wide analyses of embryonic human and mouse germlines reveal a conserved transcriptional program, initiated in PGCs after gonadal colonization, that differentiates germ cells from their germline precursors and from somatic lineages. Through genetic studies in mice and pigs, we demonstrate that one such gonad-induced factor, the RNA-binding protein DAZL, is necessary in vivo to restrict the developmental potential of the germline; DAZL's absence prolongs expression of a Nanog pluripotency reporter, facilitates derivation of pluripotent cell lines, and causes spontaneous gonadal teratomas. Based on these observations in humans, mice, and pigs, we propose that germ cells are determined after gonadal colonization in mammals. We suggest that germ cell determination was induced late in embryogenesis-after organogenesis has begun-in the common ancestor of all vertebrates, as in modern mammals, where this transition is induced by somatic cells of the gonad. We suggest that failure of this process of germ cell determination likely accounts for the origin of human testis cancer., Competing Interests: Competing interest statement: A.L.W., D.F.C., and S.C.F. are employees and shareholders of Recombinetics Inc. I.D. is a member of the scientific advisory board of Recombinetics Inc. The remaining authors have declared that no competing interests exist.

Blastocyst complementation combined with gene editing is an emerging approach in the field of regenerative medicine that could potentially solve the worldwide problem of organ shortages for transplantation. In theory, blastocyst complementation can generate fully functional human organs or tissues, grown within genetically engineered livestock animals. Targeted deletion of a specific gene(s) using gene editing to cause deficiencies in organ development can open a niche for human stem cells to occupy, thus generating human tissues. Within this review, we will focus on the pancreas, liver, heart, kidney, lung, and skeletal muscle, as well as cells of the immune and nervous systems. Within each of these organ systems, we identify and discuss (i) the common causes of organ failure; (ii) the current state of regenerative therapies; and (iii) the candidate genes to knockout and enable specific exogenous organ development via the use of blastocyst complementation. We also highlight some of the current barriers limiting the success of blastocyst complementation.

Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 ( NF1 ). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies., Competing Interests: A.L.W., T.R., A.W.C., D.F.C., S.C.F., and D.A.L. are employees and/or shareholders of Recombinetics Inc. C.L.M. is a consultant to Recombinetics Inc. The remaining authors declare no competing interests.

Drone autopilots are naturally suited for real-time iceberg tracking as they measure position and orientation (pitch, roll, and heading) and they transmit these data to a ground station. We powered an ArduPilot Mega (APM) 2.6 with a 5V 11 Ah lithium ion battery (a smartphone power bank), placed the APM and battery in a waterproof sportsman's box, and tossed the box and its contents by hand onto an 80 m-long iceberg from an 8 m boat. The data stream could be viewed on a laptop, which greatly enhanced safety while collecting conductivity/temperature/depth (CTD) profiles from the small boat in the iceberg's vicinity. The 10 s position data allowed us to compute the distance of each CTD profile to the iceberg, which is necessary to determine if a given CTD profile was collected within the iceberg's meltwater plume. The APM position data greatly reduced position uncertainty when compared to 5 min position data obtained from a Spot Trace unit. The APM functioned for over 10 h without depleting the battery. We describe the specific hardware used and the software settings necessary to use the APM as a real-time iceberg tracker. Furthermore, the methods described here apply to all Ardupilot-compatible autopilots. Given the low cost ($90) and ease of use, drone autopilots like the APM should be included as another tool for studying iceberg motion and for enhancing safety of marine operations. •Commercial off-the-shelf iceberg trackers are typically configured to record positions over relatively long intervals (months to years) and are not well-suited for short-term (hours to few days), high-frequency monitoring•Drone autopilots are cheap and provide high-frequency (>1 Hz) and real-time information about iceberg drift and orientation•Drone autopilots and ground control software can be easily adapted to studies of iceberg-ocean interactions and operational iceberg management.

We applied precise  zygote-mediated genome editing to eliminate beta-lactoglobulin (BLG), a major allergen in cows' milk. To efficiently generate LGB knockout cows, biopsied embryos were screened to transfer only appropriately modified embryos. Transfer of 13 pre-selected embryos into surrogate cows resulted in the birth of three calves, one dying shortly after birth. Deep sequencing results confirmed conversion of the genotype from wild type to the edited nine bp deletion by more than 97% in the two male calves. The third calf, a healthy female, had in addition to the expected nine bp deletion (81%), alleles with an in frame 21 bp deletion (<17%) at the target site. While her milk was free of any mature BLG, we detected low levels of a BLG variant derived from the minor deletion allele. This confirmed that the nine bp deletion genotype completely knocks out production of BLG. In addition, we showed that the LGB knockout animals are free of any TALEN-mediated off-target mutations or vector integration events using an unbiased whole genome analysis. Our study demonstrates the feasibility of generating precisely biallelically edited cattle by zygote-mediated editing for the safe production of hypoallergenic milk.

Background: Ossabaw pigs are unique miniature swine with genetic predisposition to develop metabolic syndrome and coronary atherosclerosis after extended periods receiving atherogenic diets. We have hypothesized that transgenic Ossabaw swine expressing chimp PCSK9 (proprotein convertase subtilisin-like/kexin type 9) containing the D374Y gain of function would develop familial hypercholesterolemia and coronary artery plaques more rapidly than Landrace swine with the same transgene., Methods and Results: Ossabaw and Landrace PCSK9 gain-of-function founders were generated by Sleeping Beauty transposition and cloning. Histopathologic findings in the Ossabaw founder animal showed more advanced plaques and higher stenosis than in the Landrace founder, underscoring the Ossabaw genetic predisposition to atherosclerosis. We chose to further characterize the Ossabaw PCSK9 gain-of-function animals receiving standard or atherogenic diets in a 6-month longitudinal study using computed tomography, magnetic resonance (MR) imaging, intravascular ultrasound, and optical coherence tomography, followed by pathological analysis of atherosclerosis focused on the coronary arteries. The Ossabaw model was consistently hypercholesterolemic, with or without dietary challenge, and by 6 months had consistent and diffuse fibrofatty or fibroatheromatous plaques with necrosis, overlying fibrous caps, and calcification in up to 10% of coronary plaques., Conclusions: The Ossabaw PCSK9 gain-of-function model provides consistent and robust disease development in a time frame that is practical for use in preclinical therapeutic evaluation to drive innovation. Although no animal model perfectly mimics the human condition, this genetic large-animal model is a novel tool for testing therapeutic interventions in the context of developing and advanced coronary artery disease., (© 2018 The Authors and Recombinetics. Published on behalf of the American Heart Association, Inc., by Wiley.)

Spermatogonia represent a diploid germ cell population that includes spermatogonial stem cells. In this report, we describe new methods for isolation of highly enriched porcine spermatogonia based on light scatter properties, and for targeted mutagenesis in porcine spermatogonia using nucleofection and TALENs. We optimized a nucleofection protocol to deliver TALENs specifically targeting the DMD locus in porcine spermatogonia. We also validated specific sorting of porcine spermatogonia based on light scatter properties. We were able to obtain a highly enriched germ cell population with over 90% of cells being UCH-L1 positive undifferentiated spermatogonia. After gene targeting in porcine spermatogonia, indel (insertion or deletion) mutations as a result of non-homologous end joining (NHEJ) were detected in up to 18% of transfected cells. Our report demonstrates for the first time an approach to obtain a live cell population highly enriched in undifferentiated spermatogonia from immature porcine testes, and that gene targeting can be achieved in porcine spermatogonia which will enable germ line modification., (© 2018 Wiley Periodicals, Inc.)

Hypercholesterolemia is a major risk factor for atherosclerosis. Remaining challenges in the management of atherosclerosis necessitate development of animal models that mimic human pathophysiology. We characterized a novel mutant pig model with DNA transposition of D374Y gain-of-function (GOF) cDNA of chimp proprotein convertase subtilisin/kexin type-9 (PCSK9), and tested the hypothesis that it would develop peripheral vascular remodeling and target organ injury in the kidney. Wild-type or PCSK9-GOF Ossabaw miniature pigs fed a standard or atherogenic diet (AD) (n = 7 each) were studied in vivo after 3 and 6 months of diet. Single-kidney hemodynamics and function were studied using multidetector computed tomography and kidney oxygenation by blood oxygen level-dependent magnetic resonance imaging. The renal artery was evaluated by intravascular ultrasound, aortic stiffness by multidetector computed tomography, and kidney stiffness by magnetic resonance elastography. Subsequent ex vivo studies included the renal artery endothelial function and morphology of abdominal aorta, renal, and femoral arteries by histology. Compared with wild type, PCSK9-GOF pigs had elevated cholesterol, triglyceride, and blood pressure levels at 3 and 6 months. Kidney stiffness increased in GOF groups, but aortic stiffness only in GOF-AD. Hypoxia, intrarenal fat deposition, oxidative stress, and fibrosis were observed in both GOF groups, whereas kidney function remained unchanged. Peripheral arteries in GOF groups showed medial thickening and development of atheromatous plaques. Renal endothelial function was impaired only in GOF-AD. Therefore, the PCSK9-GOF mutation induces rapid development of atherosclerosis in peripheral vessels of Ossabaw pigs, which is exacerbated by a high-cholesterol diet. This model may be useful for preclinical studies of atherosclerosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In this work we use TALE nucleases (TALENs) to target a reporter construct to the DDX4 ( vasa ) locus in chicken primordial germ cells (PGCs). Vasa is a key germ cell determinant in many animal species and is posited to control avian germ cell formation. We show that TALENs mediate homology-directed repair of the DDX4 locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire DDX4 locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce DDX4 null offspring. In DDX4 knockout chickens, PGCs are initially formed but are lost during meiosis in the developing ovary, leading to adult female sterility. TALEN-mediated gene targeting in avian PGCs is therefore an efficient process., (© 2017. Published by The Company of Biologists Ltd.)

The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was detected. The total number of genetic modifications (29) was higher than the total number of gene-edited embryos, as three blastocysts from the group RNA2X reported more than one type of modification. The modifications included indels (10/56; 17.9%) and large deletions (19/56; 33.9%). Moreover, it was possible to detect HR in 1/8 (12.5%) embryos treated with RNA2X. These results report that the CRISPR/Cas9 system can be applied for site-specific edition of the bovine genome, which could have a great impact on the development of large animals resistant to important zoonotic diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

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.

The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3(IL2Rγ) consomic model compared with the SS(IL2Rγ) parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3(IL2Rγ) background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk., (©2014 American Association for Cancer Research.)

The advent of Transcription Activator-Like Effector Nucleases (TALENs), and similar technologies such as CRISPR, provide a straightforward and cost effective option for targeted gene knockout (KO). Yet, there is still a need for methods that allow for enrichment and isolation of modified cells for genetic studies and therapeutics based on gene modified human cells. We have developed and validated two methods for simple enrichment and isolation of single or multiplex gene KO's in transformed, immortalized, and human progenitor cells. These methods rely on selection of a phenotypic change such as resistance to a particular drug or ability to grow in a selective environment. The first method, termed co-transposition, utilizes integration of a piggyBac transposon vector encoding a drug resistance gene. The second method, termed co-targeting, utilizes TALENs to KO any gene that when lost induces a selectable phenotype. Using these methods we also show removal of entire genes and demonstrate that TALENs function in human CD34+ progenitor cells. Further, co-transposition can be used to generate conditional KO cell lines utilizing an inducible cDNA rescue transposon vector. These methods allow for robust enrichment and isolation of KO cells in a rapid and efficient manner.

The Sleeping Beauty (SB) transposon is the most widely used DNA transposon in genetic applications and is the only DNA transposon thus far in clinical trials for human gene therapy. In the absence of atomic level structural information, the development of SB transposon relied primarily on the biochemical and genetic homology data. While these studies were successful and have yielded hyperactive transposases, structural information is needed to gain a mechanistic understanding of transposase activity and guides to further improvement. We have initiated a structural study of SB transposase using Nuclear Magnetic Resonance (NMR) and Circular Dichroism (CD) spectroscopy to investigate the properties of the DNA-binding domain of SB transposase in solution. We show that at physiologic salt concentrations, the SB DNA-binding domain remains mostly unstructured but its N-terminal PAI subdomain forms a compact, three-helical structure with a helix-turn-helix motif at higher concentrations of NaCl. Furthermore, we show that the full-length SB DNA-binding domain associates differently with inner and outer binding sites of the transposon DNA. We also show that the PAI subdomain of SB DNA-binding domain has a dominant role in transposase's attachment to the inverted terminal repeats of the transposon DNA. Overall, our data validate several earlier predictions and provide new insights on how SB transposase recognizes transposon DNA.

Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.

Over the past 5 years there has been a major transformation in our ability to precisely manipulate the genomes of animals. Efficiencies of introducing precise genetic alterations in large animal genomes have improved 100000-fold due to a succession of site-specific nucleases that introduce double-strand DNA breaks with a specificity of 10(-9). Herein we describe our applications of site-specific nucleases, especially transcription activator-like effector nucleases, to engineer specific alterations in the genomes of pigs and cows. We can introduce variable changes mediated by non-homologous end joining of DNA breaks to inactive genes. Alternatively, using homology-directed repair, we have introduced specific changes that support either precise alterations in a gene's encoded polypeptide, elimination of the gene or replacement by another unrelated DNA sequence. Depending on the gene and the mutation, we can achieve 10%-50% effective rates of precise mutations. Applications of the new precision genetics are extensive. Livestock now can be engineered with selected phenotypes that will augment their value and adaption to variable ecosystems. In addition, animals can be engineered to specifically mimic human diseases and disorders, which will accelerate the production of reliable drugs and devices. Moreover, animals can be engineered to become better providers of biomaterials used in the medical treatment of diseases and disorders.