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In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.

During the late nineteenth century large numbers of long-distance travelers, often elites from imperial states, ventured abroad. The purposes of these travels included for scientific or academic research, for reporting or other information-gathering purposes, and for touristic experiences. These imperial travelers' observations and experiences can be analyzed to provide us a fuller picture of imperial peripheries. Most existing studies of long-distance travel focus on individual travelers' accounts of specific journeys in isolation, rather than in a comparative framework. Comparative study of travel accounts can be used to better understand why different imperial actors supported these sorts of travels, and how written descriptions provide us with varied views of empire. Each travelers' experiences should be analyzed holistically, by investigating their biographical information, identities, class positions, and other individual characteristics, to effectively analyze the significance of their observations.

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

We report results from simultaneous experiments conducted in late 2022 in Belarus, Estonia, Kazakhstan, Russia and Ukraine. The experiments focus on fact-checking misinformation supportive of Russia in the Russia-Ukraine War. Meta-analysis makes clear that fact-checking misinformation reduces belief in pro-Kremlin false claims. Effects of fact-checks are not uniform across countries; our meta-analytic estimate is reliant on belief accuracy increases observed in Russia and Ukraine. While fact-checks improve belief accuracy, they do not change respondents’ attitudes about which side to support in the War. War does not render individuals hopelessly vulnerable to misinformation—but fact-checking misinformation is unlikely to change their views toward the conflict.

Cas9 (from Streptococcus pyogenes) in complex with a guide RNA targets complementary DNA for cleavage. Here, we developed a single-molecule FRET analysis to study the mechanisms of specificity enhancement of two engineered Cas9s (eCas9 and Cas9-HF1). A DNA-unwinding assay showed that mismatches affect cleavage reactions through rebalancing the unwinding–rewinding equilibrium. Increasing PAM-distal mismatches facilitates rewinding, and the associated cleavage impairment shows that cleavage proceeds from the unwound state. Engineered Cas9s depopulate the unwound state more readily with mismatches. The intrinsic cleavage rate is much lower for engineered Cas9s, preventing cleavage from transiently unwound off-targets. Engineered Cas9s require approximately one additional base pair match for stable binding, freeing them from sites that would otherwise sequester them. Therefore, engineered Cas9s achieve their improved specificity by inhibiting stable DNA binding to partially matching sequences, making DNA unwinding more sensitive to mismatches and slowing down the intrinsic cleavage reaction.

By coupling synoptic data from a basin-wide assessment of streamwater chemistry with network-based geostatistical analysis, we show that spatial processes differentially affect biogeochemical condition and pattern across a headwater stream network. We analyzed a high-resolution dataset consisting of 664 water samples collected every 100 m throughout 32 tributaries in an entire fifth-order stream network. These samples were analyzed for an exhaustive suite of chemical constituents. The fine grain and broad extent of this study design allowed us to quantify spatial patterns over a range of scales by using empirical semivariograms that explicitly incorporated network topology. Here, we show that spatial structure, as determined by the characteristic shape of the semivariograms, differed both among chemical constituents and by spatial relationship (flow-connected, flow-unconnected, or Euclidean). Spatial structure was apparent at either a single scale or at multiple nested scales, suggesting separate processes operating simultaneously within the stream network and surrounding terrestrial landscape. Expected patterns of spatial dependence for flow-connected relationships (e.g., increasing homogeneity with downstream distance) occurred for some chemical constituents (e.g., dissolved organic carbon, sulfate, and aluminum) but not for others (e.g., nitrate, sodium). By comparing semivariograms for the different chemical constituents and spatial relationships, we were able to separate effects on streamwater chemistry of (i) fine-scale versus broad-scale processes and (ii) in-stream processes versus landscape controls. These findings provide insight on the hierarchical scaling of local, longitudinal, and landscape processes that drive biogeochemical patterns in stream networks.

The structure of E. coli Cascade bound to foreign target DNA is presented, revealing the basis of the relaxed Cascade PAM recognition specificity, which results from its interaction with the minor groove, and demonstrating how a wedge in Cascade forces the directional pairing of the target strand with CRISPR RNA while stabilizing the non-target displaced strand. Structure of DNA-bound Cascade complex In the CRISPR system of bacterial immune surveillance, now widely used for genome editing, a CRISPR RNA (crRNA)-bound Cascade complex interacts with double-stranded DNA that can undergo complementary base pairing. The crRNA binds the target strand to form an R-loop structure. The trinucleotide PAM motif near the target sequence is responsible for non-self discrimination. Ailong Ke and colleagues have solved the structure of Cascade bound to foreign target DNA. This reveals the basis of Cascade's relaxed PAM specificity, resulting from its interaction with the minor groove, and shows how a wedge in Cascade forces the directional pairing of the target strand with crRNA, and at the same time stabilizes the non-target, displaced strand. Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes 1 . Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate 2 , 3 . During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop 4 , 5 . Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands 4 , 6 , 7 . A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination 4 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5′-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process 17 , 18 .

Previous work comparing the developmental mechanisms involved in digit reduction in horses with other mammals reported that horses have only a ‘single digit', with two flanking metapodials identified as remnants of digit II and IV. Here we show that early Equus embryos go through a stage with five digit condensations, and that the flanking splint metapodials result from fusions of the two anterior digits I and II and the two posterior digits IV and V, in a striking parallel between ontogeny and phylogeny. Given that even this most extreme case of digit reduction exhibits primary pentadactyly, we re-examined the initial stages of digit condensation of all digit-reduced tetrapods where data are available and found that in all cases, five or four digits initiate (four with digit I missing). The persistent pentadactyl initiation in the horse and other digit-reduced modern taxa underscores a durable developmental stability at the initiation of digits. The digit evodevo model may help illuminate the biological circumstances under which organ systems become highly stabilized versus highly plastic.

Tidal wetland restoration through dike removal can enhance coastal ecosystem services, such as flood attenuation, fish production, and carbon sequestration. However, landscape-level heterogeneity may influence recovery. For a 169-ha restoration project in Tillamook Bay, Oregon, we hypothesized that areas of more intensive pre-restoration land use/land cover (cropping, grazing) would differ more from reference conditions before restoration than less-intensive uses and that initial post-restoration recovery would vary by land-use/land-cover type and wetland elevation. Before the restoration, the project site overall had higher nonnative plant cover, lower elevation and groundwater levels, and lower soil pH than reference high marsh, with some differences by land-use/land-cover type. The cropped and grazed areas were strongly dominated by non-native species, such as Phalaris arundinacea, and were 74 and 31 cm lower than reference high marsh. Less intensively managed areas had elevations intermediate to the cropped and grazed areas and a trend towards higher native plant cover. The restoration led to higher dry-season groundwater levels, increased soil salinity to mesohaline conditions, and a 10-fold increase in soil pH at the project site, while reducing total plant cover. The degree of pre-to early post-restoration change for some parameters differed by land-use/landcover type (total and non-native plant cover) and by wetland elevation (soil salinity, pH, and accretion rate; and total and nonnative plant cover). Our results suggest that pre-restoration heterogeneity in elevation and land cover/land use may influence early post-restoration recovery. Restoration planning can incorporate such spatial variability into management targets and interventions for specific outcomes.