Asymmetric parental genome engineering by Cas9 during mouse meiotic exit

Mammalian genomes can be edited by injecting pronuclear embryos with Cas9 cRNA and guide RNA (gRNA) but it is unknown whether editing can also occur during the onset of embryonic development, prior to pronuclear embryogenesis. We here report Cas9-mediated editing during sperm-induced meiotic exit and the initiation of development. Injection of unfertilized, mouse metaphase II (mII) oocytes with Cas9 cRNA, gRNA and sperm enabled efficient editing of transgenic and native alleles. Pre-loading oocytes with Cas9 increased sensitivity to gRNA ~100-fold. Paternal allelic editing occurred as an early event: single embryo genome analysis revealed editing within 3 h of sperm injection, coinciding with sperm chromatin decondensation during the gamete-to-embryo transition but prior to pronucleus formation. Maternal alleles underwent editing after the first round of DNA replication, resulting in mosaicism. Asymmetric editing of maternal and paternal alleles suggests a novel strategy for discriminatory targeting of parental genomes

Switchable genome editing via genetic code expansion

Multiple applications of genome editing by CRISPR-Cas9 necessitate stringent regulation and Cas9 variants have accordingly been generated whose activity responds to small ligands, temperature or light. However, these approaches are often impracticable, for example in clinical therapeutic genome editing in situ or gene drives in which environmentally-compatible control is paramount. With this in mind, we have developed heritable Cas9-mediated mammalian genome editing that is acutely controlled by the cheap lysine derivative, Lys(Boc) (BOC). Genetic code expansion permitted non-physiological BOC incorporation such that Cas9 (Cas9BOC) was expressed in a full-length, active form in cultured somatic cells only after BOC exposure. Stringently BOC-dependent, heritable editing of transgenic and native genomic loci occurred when Cas9BOC was expressed at the onset of mouse embryonic development from cRNA or Cas9BOC transgenic females. The tightly controlled Cas9 editing system reported here promises to have broad applications and is a first step towards purposed, spatiotemporal gene drive regulation over large geographical ranges

Mice produced by mitotic reprogramming of sperm injected into haploid parthenogenotes

Sperm are highly differentiated and the activities that reprogram them for embryonic development during fertilization have historically been considered unique to the oocyte. We here challenge this view and demonstrate that mouse embryos in the mitotic cell cycle can also directly reprogram sperm for full-term development. Developmentally incompetent haploid embryos (parthenogenotes) injected with sperm developed to produce healthy offspring at up to 24% of control rates, depending when in the embryonic cell cycle injection took place. This implies that most of the first embryonic cell cycle can be bypassed in sperm genome reprogramming for full development. Remodelling of histones and genomic 5′-methylcytosine and 5′-hydroxymethylcytosine following embryo injection were distinct from remodelling in fertilization and the resulting 2-cell embryos consistently possessed abnormal transcriptomes. These studies demonstrate plasticity in the reprogramming of terminally differentiated sperm nuclei and suggest that different epigenetic pathways or kinetics can establish totipotency

Plans and Status of Wind-Tunnel Testing Employing an Aeroservoelastic Semispan Model

This paper presents the research objectives, summarizes the pre-wind-tunnel-test experimental results to date, summarizes the analytical predictions to date, and outlines the wind-tunnel-test plans for an aeroservoelastic semispan wind-tunnel model. The model is referred to as the Supersonic Semispan Transport (S4T) Active Controls Testbed (ACT) and is based on a supersonic cruise configuration. The model has three hydraulically-actuated surfaces (all-movable horizontal tail, all-movable ride control vane, and aileron) for active controls. The model is instrumented with accelerometers, unsteady pressure transducers, and strain gages and will be mounted on a 5-component sidewall balance. The model will be tested twice in the Langley Transonic Dynamics Tunnel (TDT). The first entry will be an "open-loop" model-characterization test; the second entry will be a "closed-loop" test during which active flutter suppression, gust load alleviation and ride quality control experiments will be conducted

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry

Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ∼40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive â€'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (∼96 h). 5 hmC levels were consistently low (<0. 2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes

Brangus cows have ovarian reserve parameters more like Brahman than Angus cows

Bos indicus females have more surface antral follicles than Bos taurus females; however, histological studies demonstrated no difference in total number of primordial follicles between these two biological types of cattle. Primordial follicle density in the ovary was less in Nelore ovaries compared to Angus ovaries, but no studies have examined the primordial follicle density in Bos indicus cross-bred females. It, therefore, was hypothesized that primordial follicle density in the ovary would decrease as percentage Bos indicus increased. Ovaries were collected from cross-bred Angus (n=32, no Bos indicus influence), Brangus (n=15), or Brahman (n=9) cows and prepared for histological evaluation. There was no difference in total number of primordial follicles per ovary between breeds (P \u3e 0.10). When numbers of primordial follicles were expressed on a per gram of ovarian tissue basis, there were fewer primordial follicles per gram of ovarian tissue in Brangus and Brahman cows than in Angus cows (P \u3c 0.05). Brangus cows did not differ from Brahman cows in primordial follicle density (P \u3e 0.10). Differences in primordial follicle density could indicate differences in capacity of ovarian stroma to produce factors necessary for oogonial proliferation and primordial follicle formation among breeds. Identifying these factors could improve the aprroach for culturing pre-antral follicles of cattle. Furthermore, these results explain why ultrasonographic antral follicle counts may need to be adjusted to a greater threshold to predict size of the ovarian reserve and determine ovarian reserve related reproductive traits in Bos indicus females

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

ASSESSING TARGET SPECIFICITY OF THE SMALL MOLECULE INHIBITOR MARIMASTAT TO SNAKE VENOM TOXINS: A NOVEL APPLICATION OF THERMAL PROTEOME PROFILING

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation, and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including Thermal Proteome Profiling (TPP) and Proteome Integral Solubility Alteration (PISA) assays, represent “deep proteomics” methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply TPP and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom