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Abstract Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals – Mg²⁺, Ca²⁺, and Co²⁺– promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9’s metal-dependent activity. The ionic strength thereby promotes Cas9’s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms. 

Abstract CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction^1–5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes^6–8. Here, we determine the structures of theSynechocystistype III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors^9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2′-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes. 

Bioremediation is the use of microorganisms for the degradation or removal of contaminants. Most bioremediation research has focused on processes performed by the domain Bacteria ; however, Archaea are known to play important roles in many situations. In extreme conditions, such as halophilic or acidophilic environments, Archaea are well suited for bioremediation. In other conditions, Archaea collaboratively work alongside Bacteria during biodegradation. In this review, the various roles that Archaea have in bioremediation is covered, including halophilic hydrocarbon degradation, acidophilic hydrocarbon degradation, hydrocarbon degradation in nonextreme environments such as soils and oceans, metal remediation, acid mine drainage, and dehalogenation. Research needs are addressed in these areas. Beyond bioremediation, these processes are important for wastewater treatment (particularly industrial wastewater treatment) and help in the understanding of the natural microbial ecology of several Archaea genera. 

Abstract BackgroundThe stable fly,Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies. ResultsThis study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways. ConclusionsThe combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship ofStomoxysto other blood-feeding (horn flies andGlossina) and non-blood-feeding flies (house flies, medflies,Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.