Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes.

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14, and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for DNA cleavage inhibition and have divergent C termini that are required in each case for inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-AcrIIA15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-AcrIIA15 as unique bifunctional inhibitors of SauCas9

Variation in small sapling density, understory cover, and resource availability in four neotropical forests

Even though many forest plants spend all or a significant portion of their lives in the forest understoty, few studies have compared understory composition, structure, and resource availability among forests. We used standardized transect-based methods to compare small sapling densities (10-50 cm tall), understory vegetation cover, canopy openness, and nutrient availability in non-gap portions of four lowland Neotropical forests: La Selva, Costa Rica (LS), Barro Colorado Island, Panama (BCI), Cocha Cashu, Peru (CC), and north of Manaus, Brazil (KM41). Sites differed significantly in all variables except canopy openness. LS had high palm and non-fern herb cover and low density of small saplings (0.7-1.6/m2) compared to other sites. CC had high fern cover, whereas BCI had low cover in all categories of understory vegetation (palms, ferns, and non-fern herbaceous plants). BCI, CC, and KM41 had similar small sapling densities, ranging from 4.8-7.5/m 2. Within each forest, cation (Ca, Mg, K, and Na) availability was usually higher on more fertile soil orders (Inceptisols, Alfisols, and Entisols) than on more weathered soil types (Ultisols and Oxisols). Extractable P was highest at LS and CC and lowest on BCI (no data for KM41). Spatial autocorrelation was present for some variables in some transects to distances beyond our detection ability (\u3e25 m). Understory palm cover was negatively correlated with small sapling density at fine (1 m2 quadrat) and coarse spatial scales (among forests), although across forests the effect of palms was due entirely to the difference between LS and the other three forests. These results provide cross-site support for the hypothesis that understory cover by palms decreases the density of small saplings that comprise the advance regeneration of the forest

Synthesis, Electrochemistry, and Excited-State Properties of Three Ru(II) Quaterpyridine Complexes

The complexes [Ru(qpy)LL′]2+ (qpy = 2,2′:6′,2″:6″,2‴-quaterpyridine), with 1: L = acetonitrile, L′= chloride; 2: L = L′= acetonitrile; and 3: L = L′= vinylpyridine, have been prepared from [Ru(qpy) (Cl)2]. Their absorption spectra in CH3CN exhibit broad metal-to-ligand charge transfer (MLCT) absorptions arising from overlapping 1A1 → 1MLCT transitions. Photoluminescence is not observed at room temperature, but all three are weakly emissive in 4:1 ethanol/methanol glasses at 77 K with broad, featureless emissions observed between 600 and 1000 nm consistent with MLCT phosphorescence. Cyclic voltammograms in CH3CN reveal the expected RuIII/II redox couples. In 0.1 M trifluoroacetic acid (TFA), 1 and 2 undergo aquation to give [RuII(qpy)(OH2)2]2+, as evidenced by the appearance of waves for the couples [RuIII(qpy)(OH2)2]3+/[RuII(qpy)(OH2)2]2+, [RuIV(qpy)(O)(OH2)]2+/[RuIII(qpy)(OH2)2]3+, and [RuVI(qpy)(O)2]2+/[RuIV(qpy)(O)(OH2)]2+ in cyclic voltammograms

Differentiation of primate primordial germ cell-like cells following transplantation into the adult gonadal niche.

A major challenge in stem cell differentiation is the availability of bioassays to prove cell types generated in vitro are equivalent to cells in vivo. In the mouse, differentiation of primordial germ cell-like cells (PGCLCs) from pluripotent cells was validated by transplantation, leading to the generation of spermatogenesis and to the birth of offspring. Here we report the use of xenotransplantation (monkey to mouse) and homologous transplantation (monkey to monkey) to validate our in vitro protocol for differentiating male rhesus (r) macaque PGCLCs (rPGCLCs) from induced pluripotent stem cells (riPSCs). Specifically, transplantation of aggregates containing rPGCLCs into mouse and nonhuman primate testicles overcomes a major bottleneck in rPGCLC differentiation. These findings suggest that immature rPGCLCs once transplanted into an adult gonadal niche commit to differentiate towards late rPGCs that initiate epigenetic reprogramming but do not complete the conversion into ENO2-positive spermatogonia

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders