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.

Does a Rising Median Income Lift All Birth Weights? County Median Income Changes and Low Birth Weight Rates Among Births to Black and White Mothers.

Policy Points Policies that increase county income levels, particularly for middle-income households, may reduce low birth weight rates and shrink disparities between Black and White infants. Given the role of aggregate maternal characteristics in predicting low birth weight rates, policies that increase human capital investments (e.g., funding for higher education, job training) could lead to higher income levels while improving population birth outcomes. The association between county income levels and racial disparities in low birth weight is independent of disparities in maternal risks, and thus a broad set of policies aimed at increasing income levels (e.g., income supplements, labor protections) may be warranted. CONTEXT: Low birth weight (LBW; <2,500 grams) and infant mortality rates vary among place and racial group in the United States, with economic resources being a likely fundamental contributor to these disparities. The goals of this study were to examine time-varying county median income as a predictor of LBW rates and Black-White LBW disparities and to test county prevalence and racial disparities in maternal sociodemographic and health risk factors as mediators. METHODS: Using national birth records for 1992-2014 from the National Center for Health Statistics, a total of approximately 27.4 million singleton births to non-Hispanic Black and White mothers were included. Data were aggregated in three-year county-period observations for 868 US counties meeting eligibility requirements (n = 3,723 observations). Sociodemographic factors included rates of low maternal education, nonmarital childbearing, teenage pregnancy, and advanced-age pregnancy; and health factors included rates of smoking during pregnancy and inadequate prenatal care. Among other covariates, linear models included county and period fixed effects and unemployment, poverty, and income inequality. FINDINGS: An increase of $10,000 in county median income was associated with 0.34 fewer LBW cases per 100 live births and smaller Black-White LBW disparities of 0.58 per 100 births. Time-varying county rates of maternal sociodemographic and health risks mediated the association between median income and LBW, accounting for 65% and 25% of this estimate, respectively, but racial disparities in risk factors did not mediate the income association with Black-White LBW disparities. Similarly, county median income was associated with very low birth weight rates and related Black-White disparities. CONCLUSIONS: Efforts to increase income levels-for example, through investing in human capital, enacting labor union protections, or attracting well-paying employment-have broad potential to influence population reproductive health. Higher income levels may reduce LBW rates and lead to more equitable outcomes between Black and White mothers.

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.

Measurement and control of optical nonlinearities in dispersive dielectric multilayers.

Dispersive dielectric multilayer mirrors, high-dispersion chirped mirrors in particular, are widely used in modern ultrafast optics to manipulate spectral chirps of ultrashort laser pulses. Dispersive mirrors are routinely designed for dispersion compensation in ultrafast lasers and are assumed to be linear optical components. In this work, we report the experimental characterization of an unexpectedly strong nonlinear response in these chirped mirrors. At modest peak intensities <2 TW/cm2-well below the known laser-induced damage threshold of these dielectric structures-we observed a strong reflectivity decrease, local heating, transient spectral modifications, and time-dependent absorption of the incident pulse. Through computational analysis, we found that the incident laser field can be enhanced by an order of magnitude in the dielectric layers of the structure. The field enhancement leads to a wavelength-dependent nonlinear absorption, that shows no signs of cumulative damage before catastrophic failure. The nonlinear absorption is not a simply two-photon process but instead is likely mediated by defects that facilitate two-photon absorption. To mitigate this issue, we designed and fabricated a dispersive multilayer design that strategically suppresses the field enhancement in the high-index layers, shifting the high-field regions to the larger-bandgap, low-index layers. This strategy significantly increases the maximum peak intensity that the mirror can sustain. However, our finding of an onset of nonlinear absorption even at 'modest' fluence and peak intensity has significant implications for numerous past published experimental works employing dispersive mirrors. Additionally, our results will guide future ultrafast experimental work and ultrafast laser design.

The Effectiveness of Shared Compared to Informed Decision Making for Prostate Cancer Screening in a High-Risk African American Population: A Randomized Control Trial.

BACKGROUND: Prostate cancer incidence and mortality in the United States in African Americans (AA) are higher than in Caucasians. Eastern Cuyahoga County in Ohio is majority AA and is considered an underserved population particularly vulnerable to healthcare disparities. There is a paucity of data about shared decision making among high-risk AA men with regard to prostate cancer screening. This study aims to examine shared versus informed decision making (SDM versus IDM) in a randomized, control trial among a large, high-risk AA population. METHODS: Patients were included in annual one-day outreach events, each held over 3 years (2017-2019), and were randomized at each event into IDM (control) and SDM (investigational) groups and then were offered screening via prostate specific antigen (PSA) and digital rectal exam (DRE). The primary endpoints were proportion of participants over 40 who did not demonstrate decisional conflict about prostate cancer screening measured by the SURE score, as well as change of knowledge score about prostate cancer screening. RESULTS: Overall, 175 patients were enrolled in the trial; 79 in the SDM arm and 96 in the IDM arm. The investigational (SDM) arm had 3/79 (3.9%) conflict versus 6/96 (6.4%) in the control (IDM) arm (p = 0.74). With regard to knowledge improvement, the SDM cohort demonstrated improvement following educational tools for 66/79 (81%) of participants versus 76/96 (79%) in the IDM cohort (p = 0.85). There was no difference in the proportion (63%) of participants in either group who found the information very helpful (using a Likert scale). CONCLUSIONS: Our education-based study showed no significant difference between SDM and IDM with regard to decisional conflict about prostate cancer screening. The study also demonstrated significant improvement in knowledge about prostate cancer screening in a high-risk AA population in both groups. Our results should be interpreted with caution due to several limitations; however, the study can serve as a benchmark for future studies in this very important topic.

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.

Electrocardiographic predictors of pacemaker battery depletion: Diagnostic sensitivity, specificity, and clinical risk.

BACKGROUND: Pacemaker battery depletion triggers alert for replacement notification and results in automatic reprogramming, which has been shown to be associated with relevant cardiorespiratory symptoms and adverse clinical events. OBJECTIVE: Determine if electrocardiogram (ECG) pacing features may be predictive of pacemaker battery depletion and clinical risk. METHODS: This is an ECG substudy of a cohort analysis of 298 subjects referred for pacemaker generator replacement from 2006 to 2017. Electronic medical record review was performed; clinical, ECG, and pacemaker characteristics were abstracted. We applied two ECG prediction rules for pacemaker battery depletion that are relevant to all major pacemaker manufacturers except Boston Scientific and MicroPort: (1) atrial pacing not at a multiple of 10 and (2) nonsynchronous ventricular pacing not at a multiple of 10, to determine diagnostic sensitivity, specificity, and risk in applicable ECG subjects. RESULTS: We excluded 32 subjects not at replacement notification or duplicate surgeries. Overall, 176 of 266 subjects (66.2%) demonstrated atrial pacing or nonsynchronous ventricular pacing on preoperative ECG. When utilizing both rules, 139 of 176 preoperative ECGs and 12 of 163 postoperative ECGs met criteria for battery depletion yielding reasonable sensitivity (79.0%), high specificity (92.6%), and a positive likelihood ratio of 11.6:1. These rules were associated with significant increase in cardiorespiratory symptoms (P < .001) and adverse clinical events (P < .025). CONCLUSIONS: The "Rules of Ten" provided reasonable sensitivity and specificity for detecting replacement notification in pacemaker subjects with an applicable ECG. This ECG tool may help clinicians identify most patients with pacemaker battery depletion at significant clinical risk.

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.

The extraordinary optical transmission and sensing properties of Ag/Ti composite nanohole arrays.

Ag/Ti composite nanohole arrays are fabricated by a simple combination of nanosphere lithography, reactive ion etching, and dual e-beam deposition techniques. Based on the X-ray diffraction, conductivity, and ellipsometry measurements, there exists a Ag composition threshold CAg = 80 at% above which Ag is percolated through the entire film. Significant extraordinary optical transmission (EOT) is observed in nanohole samples with composition larger than the threshold. The main EOT peak position, the (1,0) Ag/glass resonance peak, redshifts as the CAg value decreased, but its index sensitivity monotonically increased with CAg till a value of 300 RIU nm-1 for the CAg = 100 at% sample is achieved. However, the LSPR peak of the nanoholes can achieve a sensitivity of 390 nm RIU-1 when CAg decreases from 100 at% to 85 at%. This study demonstrates that besides the shape, size, and measurement configuration, the resonances and sensitivities of nanohole arrays can be effectively predicted and tuned by the composition of a plasmonic composite.

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.

The Symptoms and Clinical events associated with Automatic Reprogramming (SCARE) at replacement notification study.

BACKGROUND: Pacemaker patients experience battery depletion that activates pacemaker's alert for replacement notification. Automatic reprogramming at replacement notification can result in loss of rate response and atrioventricular (AV) synchrony. OBJECTIVE: To determine if relevant symptoms or clinical events may be associated with automatic reprogramming at replacement notification. METHODS: Electronic medical record review was undertaken for 298 patients referred for pacemaker generator replacement. Primary endpoints were symptoms or clinical events during replacement notification period. RESULTS: Following elimination of duplicate pacemaker replacements (n = 12), "near-replacement notification" or "recalled" (n = 15) and pacemakers at "end of life" (n = 5), 266 subjects were included. Three distinct reprogramming cohorts were identified; those with no change (control) in pacing mode (n = 46), those with loss of rate response (n = 154), and those with loss of AV synchrony ± rate response (n = 66). In total, 83 subjects (31.2%) had symptoms with significant differences seen between groups (control = 4.3%, loss of rate response = 26.0%, loss of AV synchrony ± rate response = 62.1%, P < 0.001). Overall, 28 subjects (10.5%) experienced clinical events with significant differences seen between groups (control = 0.0%, loss of rate response = 6.5%, loss of AV synchrony ± rate response = 27.3%, P < 0.001). CONCLUSIONS: Automatic reprogramming at replacement notification was associated with significant symptoms in 26% of those who lost rate response and in 62% of those who lost AV synchrony ± rate response. Additionally, 27% of the latter cohort required nonelective clinical care.

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.

Socioeconomic variation in the association of marriage with depressive symptoms.

Although the health-relevant resources that marriage is argued to provide vary by socioeconomic status (SES), little research has examined whether the association of marriage with psychological well-being varies by SES. Focusing on depressive symptoms as an outcome and using a two-stage Heckit procedure with multilevel modeling, results from analyses of four waves of data (n = 4340 person-waves) from the American Changing Lives Survey (ACL) shows that differences in depressive symptoms between never-married and married adults varies by adjusted household income. Depressive symptoms are highest among the never married, and differences from the married greatest, at the lowest levels of income. As income increases these differences are eliminated. The conditioning effect of income is partially mediated by financial security, self-efficacy, and social support from friends and relatives. The implications of these findings for U.S. marriage promotion policies are discussed.

Ag-Cu mixed phase plasmonic nanostructures fabricated by shadow nanosphere lithography and glancing angle co-deposition.

By combining shadow nanosphere lithography with a glancing angle co-deposition technique, mixed-phase Ag-Cu triangular nanopatterns and films were fabricated. They were prepared at different compositions with respect to Ag from 100% to 0% by changing the relative deposition ratio of each metal. Characterizations by ellipsometry, energy dispersive x-ray spectroscopy, and x-ray diffraction revealed that the thin films and nanopatterns were composed of small, well-mixed Ag and Cu nano-grains with a diameter less than 20 nm, and their optical properties could be described by an effective medium theory. All compositions of the nanopattern had the same shape, but showed tunable localized surface plasmon resonance (LSPR) properties. In general, the LSPR of the nanopatterns redshifted with decreasing composition. Such a relation could be fitted by an empirical model based on the bulk theory of alloy plasmonics. By changing the colloidal template and the material deposited, this fabrication technique can be used to produce other alloy plasmonic nanostructures with predicted LSPR wavelengths.

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.