RESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.
Assuntos
Sistemas CRISPR-Cas/efeitos dos fármacos , Edição de Genes/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas Virais/genética , Vírus/genética , Archaea/genética , Archaea/imunologia , Archaea/virologia , Bactérias/genética , Bactérias/imunologia , Bactérias/virologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coevolução Biológica , Proteínas Associadas a CRISPR/antagonistas & inibidores , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA/antagonistas & inibidores , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA/efeitos dos fármacos , Endodesoxirribonucleases/antagonistas & inibidores , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Modelos Moleculares , Família Multigênica , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Vírus/metabolismo , Vírus/patogenicidadeRESUMO
In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications1. Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms2-4. Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications.
Assuntos
Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Substâncias Macromoleculares , Proteínas Virais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/química , Modelos Moleculares , Ligação Proteica , Subunidades Proteicas/metabolismo , Subunidades Proteicas/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Biotecnologia/tendências , Bacteriófagos , Proteínas Virais/metabolismoRESUMO
Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex.
Assuntos
Sistemas CRISPR-Cas , DNA/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , DNA/química , DNA de Cadeia Simples/metabolismo , Mutagênese , Ligação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismoRESUMO
The development of efficient and stable noble-metal-free electrocatalysts for hydrogen evolution reaction (HER) in alkaline media is still a challenge. Herein, a hybrid material formed by the interconnection of Ni17 W3 intermetallic compound with metallic W is demonstrated for HER. The Ni17 W3 -W hybrid is prepared by the atmosphere- and thermal-induced phase-separation strategy from a single-phase precursor (NiWO4 ), which gives Ni17 W3 -W hybrid abundant and tight interfaces. The theoretical calculation manifests that Ni17 W3 shows more optimized energetics for adsorbed H atom, while W has lower energy barrier for water dissociation, and the synergistic effect between them is believed to facilitate the HER kinetics. Moreover, Ni17 W3 presents a proper adsorption strength for both adsorbed OH and H, and thus Ni17 W3 may also act as a high HER catalyst by itself. As a result, the Ni17 W3 -W hybrid demonstrates high activity and durability for HER in liquid alkaline electrolyte; the electrolyzer assembled by Ni17 W3 -W hybrid and Ni-Fe-layered double hydroxide (LDH) as, respectively, the cathode and anode electrocatalysts presents superior performance to Pt/C-IrO2 benchmark. In addition, the Ni17 W3 -W hybrid also works well in the water electrolyzer based on solid hydroxide exchange membrane. The present work provides a promising pathway to the design of high-performance electrocatalysts.
RESUMO
The design and development of new large-capacity and selective materials for extracting rare precious metals via electronic waste is practically essential. In this paper, a new efficient UiO-66-NCS has been obtained as a consequence of the modification of the classical Zr-MOF (UiO-66-NH2), and its ability to recover gold has been investigated. These batch results adequately illustrated that UiO-66-NCS exhibited good adsorption capacity (675.5 mg g-1) and exceptional selectivity. In addition, UiO-66-NCS achieved faster adsorption equilibrium times of about 120 min. Adsorption kinetics and isotherms demonstrated that the pseudo-second-order adsorption scheme and a Langmuir-type procedure were shown by the adsorption of Au(III) on UiO-66-NCS. Characterized by pH effect experiments, TEM, XRD, and XPS, the adsorption of UiO-66-NCS with Au(III) relies on coordination, which further results in reduction, and the generated Au(0) is uniformly dispersed in the MOF. The adsorbent has considerable advantages for cyclic regeneration. Finally, DFT fitting results showed that the adsorption binding energy of UiO-66-NCS with [AuCl4]- was -8.63 kcal mol-1 for the adsorption process. UiO-66-NCS is likely to be an ideal substance for gold recovery.
RESUMO
The construction of heterostructures is an effective strategy to enhance electrocatalysis for hydrogen evolution reactions (HERs) and biomass oxidative upgrading. In this work, a Ni/TiO2 heterostructure prepared by a phase-separation strategy was adopted as a bifunctional electrocatalyst for HERs and biomass oxidation in alkaline media. Due to the optimized hydrogen adsorption energetics as well as the interfacial water structure and hydrogen bond connectivity in the electrical double layer, Ni/TiO2 exhibited high activity for HERs with an overpotential of 28 mV at 10 mA cm-2 and good durability at 1000 mA cm-2 for over 100 h in an anion exchange membrane (AEM) electrolyzer. In addition, Ni/TiO2 showed high catalytic performance for the oxidation of biomass-based platform compound 5-hydroxymethylfurfural (HMF) to high-value added compound 2,5-furandicarboxylic acid (FDCA). Continuous production of FDCA with a yield >95% was achieved in the AEM electrolyzer for over 50 h. The superior HMF oxidation performance on the Ni/TiO2 heterostructure compared to Ni resulted from stronger HMF adsorption, lower Ni3+-O formation potential, longer Ni3+-O bond and smaller Ni crystal size.
RESUMO
The electrochemical transformation of biomass to high value-added products is attractive. Herein, Cu sulfide-mediated in-situ synthesis of Cu oxide was achieved for efficient electro-oxidation of biomass derived 5-hydroxymethylfurfural (HMF) to 2,5-furandicarboxylic acid (FDCA). The copper foam-supported Cu sulfide (Cu-S/CF) was in-situ converted to Cu oxide during the electro-oxidation process. The in-situ formed Cu oxide presented high HMF conversion, FDCA yield, and faradaic efficiency in 1 m KOH with HMF concentration up to 100â mm. The oxidation of HMF on Cu oxide started with the formation of high-valence Cu species with the assistance of OH- , which then oxidized HMF spontaneously. An anion exchange membrane (AEM) electrolyzer with Cu-S/CF as the anode was assembled to continuously produce FDCA with H2 generation at the cathode. The AEM electrolyzer ran stably for 60â h with FDCA content higher than 85 % at a cell voltage between 1.50 and 1.60â V.
Assuntos
Óxidos , SulfetosRESUMO
It is a great challenge to fabricate electrode with simultaneous high activity for the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER). Herein, a high-performance bifunctional electrode formed by vertically depositing a porous nanoplate array on the surface of nickel foam is provided, where the nanoplate is made up by the interconnection of trinary Ni-Fe-Mo suboxides and Ni nanoparticles. The amorphous Ni-Fe-Mo suboxide and its in situ transformed amorphous Ni-Fe-Mo (oxy)hydroxide acts as the main active species for HER and OER, respectively. The conductive network built by Ni nanoparticles provides rapid electron transfer to active sites. Moreover, the hydrophilic and aerophobic electrode surface together with the hierarchical pore structure facilitate mass transfer. The corresponding water electrolyzer demonstrates low cell voltage (1.50 V @ 10 mA cm-2 and 1.63 V @ 100 mA cm-2) with high durability at 500 mA cm-2 for at least 100 h in 1 m KOH.
RESUMO
Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
Assuntos
Bactérias/metabolismo , Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , DNA/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bactérias/genética , Bactérias/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Microscopia Crioeletrônica , DNA/química , DNA/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Proteus penneri/genética , Proteus penneri/metabolismo , Proteus penneri/virologia , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The development of high-performance electrocatalyst with earth-abundant elements for water-splitting is a key factor to improve its cost efficiency. Herein, a noble metal-free bifunctional electrocatalyst was synthesized by a facile pyrolysis method using sucrose, urea, Co(NO3)2 and sulfur powder as raw materials. During the fabrication process, Co, S co-doped graphitic carbon nitride (g-C3N4) was first produced, and then this in-situ-formed template further induced the generation of a Co, N, S tri-doped graphene coupled with Co nanoparticles (NPs) in the following pyrolysis process. The effect of pyrolysis temperature (700, 800, and 900 °C) on the physical properties and electrochemical performances of the final product was studied. Thanks to the increased number of graphene layer encapsulated Co NPs, higher graphitization degree of carbon matrix and the existence of hierarchical macro/meso pores, the composite electrocatalyst prepared under 900 °C presented the best activity for both hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) with outstanding long-term durability. This work presented a facile method for the fabrication of non-noble-metal-based carbon composite from in-situ-formed template and also demonstrated a potential bifunctional electrocatalyst for the future investigation and application.