Efficient TALEN-mediated gene knockout in livestock Carlson, Daniel F; Tan, Wenfang; Lillico, Simon G ...
Proceedings of the National Academy of Sciences - PNAS,
10/2012, Letnik:
109, Številka:
43
Journal Article
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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.
The DNA binding domain of Transcription Activator-Like (TAL) effectors can easily be engineered to have new DNA sequence specificities. Consequently, engineered TAL effector proteins have become ...important reagents for manipulating genomes in vivo. DNA binding by TAL effectors is mediated by arrays of 34 amino acid repeats. In each repeat, one of two amino acids (repeat variable di-residues, RVDs) contacts a base in the DNA target. RVDs with specificity for C, T and A have been described; however, among RVDs that target G, the RVD NN also binds A, and NK is rare among naturally occurring TAL effectors. Here we show that TAL effector nucleases (TALENs) made with NK to specify G have less activity than their NN-containing counterparts: fourteen of fifteen TALEN pairs made with NN showed more activity in a yeast recombination assay than otherwise identical TALENs made with NK. Activity was assayed for three of these TALEN pairs in human cells, and the results paralleled the yeast data. The in vivo data is explained by in vitro measurements of binding affinity demonstrating that NK-containing TAL effectors have less affinity for targets with G than their NN-containing counterparts. On targets for which G was substituted with A, higher G-specificity was observed for NK-containing TALENs. TALENs with different N- and C-terminal truncations were also tested on targets that differed in the length of the spacer between the two TALEN binding sites. TALENs with C-termini of either 63 or 231 amino acids after the repeat array cleaved targets across a broad range of spacer lengths - from 14 to 33 bp. TALENs with only 18 aa after the repeat array, however, showed a clear optimum for spacers of 13 to 16 bp. The data presented here provide useful guidelines for increasing the specificity and activity of engineered TAL effector proteins.
Two major episodes of seagrass mortality have occurred in Florida Bay in the past 30 years: The first occurred between 1987 and 1991 and the second began in 2015. In both episodes, dense beds of ...turtle grass (Thalassia testudinum) were decimated. Elevated water temperature and hypersalinity have been implicated as contributing factors in both mortality events. During both die-off events seagrass beds on shallow (Z≈0.3 m) mudbanks in western Florida Bay were disproportionately affected when compared to neighboring seagrass in deeper basins (Z≈1.5 m). A network of in situ monitoring stations has measured sea surface temperature and salinity at hourly intervals in 16 basins in Florida Bay from August 2009 to the present day and these data aided in diagnosing the 2015–2016 die-off event. However, very few in situ observations of sea surface temperature have been collected on Florida Bay's shallow mudbanks as most are inaccessible by boat. As a result, our understanding of the role of elevated SST in shaping the spatial patterns of seagrass mortality is hindered by the spatial distribution of the in situ monitoring data and its record length. Therefore, we turn to remotely sensed SST data to expand our spatial coverage to include the entire Florida Bay ecosystem and to extend the record length to include the 1987–1991 die-off event. 1 km MODIS SST shows that shallow mudbanks were consistently warmer (by up to 6°C) than nearby deeper basins. While water depth is likely the primary driver of spatial variability in SST, Landsat-8 surface reflectance data suggest that shallow seagrass beds could have suffered from the added influence of low surface reflectance, which might have further contributed to their thermal stress. Daily 0.25° AVHRR SST shows that the August maximum SST has increased by 1°C from 1981 to 2016, which is a cause for concern for the future of seagrass in Florida Bay. Correlation of monthly AVHRR SST anomalies with the multivariate ENSO index shows that ENSO can only partially explain the anomalous temperatures. When viewed together, the in situ and remotely sensed SST suggests that both extended exposure to anomalously warm temperatures and large, rapid changes in temperature could have contributed to seagrass mortality during both events.
•Hyperthermia likely cause in two large-scale seagrass die off events.•Long-term AVHRR SST reveals increase in August temperature of 0.03°/year.•MODIS SST reveals warmer temperatures in dense seagrass beds.•Future of seagrass in Florida Bay under projected warming scenarios depends not only on magnitude of warming, but also on bathymetry and density of seagrass.
In this work we use TALE nucleases (TALENs) to target a reporter construct to the
(
) 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
locus on the Z sex chromosome at high (8.1%) efficiencies. Large genetic deletions of 30 kb encompassing the entire
locus were also created using a single TALEN pair. The targeted PGCs were germline competent and were used to produce
null offspring. In
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.
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.
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.
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.
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.
The stratification in the Northern Gulf of Eilat/Aqaba follows a well-known annual cycle of well-mixed conditions in winter, surface warming in spring and summer, maximum vertical temperature ...gradient in late summer, and erosion of stratification in fall. The strength and structure of the stratification influences the diverse coral reef ecosystem and also affects the strength of the semi-diurnal tidal currents. Long-term (13 months) moored thermistor data, combined with high temporal and vertical resolution density profiles in deep water, show that transitions from summer to fall and winter to spring/summer occur in unpredictable, pulses and are not slow and gradual, as previously deduced from monthly hydrographic measurements and numerical simulations forced by monthly climatologies. The cooling and deepening of the surface layer in fall is marked by a transition to large amplitude, semi-diurnal isotherm displacements in the stratified intermediate layer. Stratification is rebuilt in spring and summer by intermittent pulses of warm, buoyant water that can increase the upper 100–150m by 2°C that force surface waters down 100–150m over a matter of days. The stratification also varies in response to short-lived eddies and diurnal motions during winter. Thus, the variability in the stratification exhibits strong depth and seasonal dependence and occurs over range of timescales: from tidal to seasonal. We show that monthly or weekly single-cast hydrographic data under-samples the variability of the stratification in the Gulf and we estimate the error associated with single-cast assessments of the stratification.
•The majority of stratification variability occurs on timescales of 10 days or less.•Seasonal transitions are not gradual but marked by large temperature variability.•Large isotherm displacements are driven by internal tide and diurnal motions.•Spring advected heat pulses can displace surface waters and disrupt internal tide.
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