The NIH Somatic Cell Genome Editing program.

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.

Mammalian germ cells are determined after PGC colonization of the nascent gonad.

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.

Editing Myosin VB Gene to Create Porcine Model of Microvillus Inclusion Disease, With Microvillus-Lined Inclusions and Alterations in Sodium Transporters.

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.

Efficient TALEN-mediated gene targeting of chicken primordial germ cells.

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.

A reduction of primary cilia but not hedgehog signaling disrupts morphogenesis in testicular organoids.

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.

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development.

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.

Maker Buoy Variants for Water Level Monitoring and Tracking Drifting Objects in Remote Areas of Greenland.

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.

TALEN-mediated gene targeting in porcine spermatogonia.

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.

In vivo genome editing using a high-efficiency TALEN system.

The zebrafish (Danio rerio) is increasingly being used to study basic vertebrate biology and human disease with a rich array of in vivo genetic and molecular tools. However, the inability to readily modify the genome in a targeted fashion has been a bottleneck in the field. Here we show that improvements in artificial transcription activator-like effector nucleases (TALENs) provide a powerful new approach for targeted zebrafish genome editing and functional genomic applications. Using the GoldyTALEN modified scaffold and zebrafish delivery system, we show that this enhanced TALEN toolkit has a high efficiency in inducing locus-specific DNA breaks in somatic and germline tissues. At some loci, this efficacy approaches 100%, including biallelic conversion in somatic tissues that mimics phenotypes seen using morpholino-based targeted gene knockdowns. With this updated TALEN system, we successfully used single-stranded DNA oligonucleotides to precisely modify sequences at predefined locations in the zebrafish genome through homology-directed repair, including the introduction of a custom-designed EcoRV site and a modified loxP (mloxP) sequence into somatic tissue in vivo. We further show successful germline transmission of both EcoRV and mloxP engineered chromosomes. This combined approach offers the potential to model genetic variation as well as to generate targeted conditional alleles.

Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

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.

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.

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.

TALEN-based gene correction for epidermolysis bullosa.

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.

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection.

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.

Editing livestock genomes with site-specific nucleases.

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.

Compact and modular autonomous surface vehicle for water research: The Naval Operating Research Drone Assessing Climate Change (NORDACC).

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.

Macroalgal habitats support a sustained flux of floating biomass but limited carbon export beyond a Greenland fjord.

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.

Mapping intertidal macrophytes in fjords in Southwest Greenland using Sentinel-2 imagery.

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 km2 of exposed intertidal macrophytes were identified in the study area at low tide. Floating mats of macrophytes ranged in area from 400 m2 to 326,800 m2 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.

First gene-edited calf with reduced susceptibility to a major viral pathogen.

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.

DAZL Knockout Pigs as Recipients for Spermatogonial Stem Cell Transplantation.

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.