Structural insights into the diversity and DNA cleavage mechanism of Fanzor.

Fanzor (Fz) is an ωRNA-guided endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Chimeric CRISPR-CasX enzymes and guide RNAs for improved genome editing activity.

A compact protein with a size of <1,000 amino acids, the CRISPR-associated protein CasX is a fundamentally distinct RNA-guided nuclease when compared to Cas9 and Cas12a. Although it can induce RNA-guided genome editing in mammalian cells, the activity of CasX is less robust than that of the widely used S. pyogenes Cas9. Here, we show that structural features of two CasX homologs and their guide RNAs affect the R-loop complex assembly and DNA cleavage activity. Cryo-EM-based structural engineering of either the CasX protein or the guide RNA produced two new CasX genome editors (DpbCasX-R3-v2 and PlmCasX-R1-v2) with significantly improved DNA manipulation efficacy. These results advance both the mechanistic understanding of CasX and its application as a genome-editing tool.

To incise or not and where: SET-domain methyltransferases know.

The concept of the histone code posits that histone modifications regulate gene functions once interpreted by epigenetic readers. A well-studied case is trimethylation of lysine 4 of histone H3 (H3K4me3), which is enriched at gene promoters. However, H3K4me3 marks are not needed for the expression of most genes, suggesting extra roles, such as influencing the 3D genome architecture. Here, we highlight an intriguing analogy between the H3K4me3-dependent induction of double-strand breaks in several recombination events and the impact of this same mark on DNA incisions for the repair of bulky lesions. We propose that Su(var)3-9, Enhancer-of-zeste and Trithorax (SET)-domain methyltransferases generate H3K4me3 to guide nucleases into chromatin spaces, the favorable accessibility of which ensures that DNA break intermediates are readily processed, thereby safeguarding genome stability.

Mechanistic Insights into the cis- and trans-Acting DNase Activities of Cas12a.

CRISPR-Cas12a (Cpf1) is an RNA-guided DNA-cutting nuclease that has been repurposed for genome editing. Upon target DNA binding, Cas12a cleaves both the target DNA in cis and non-target single-stranded DNAs (ssDNAs) in trans. To elucidate the molecular basis for both DNase cleavage modes, we performed structural and biochemical studies on Francisella novicida Cas12a. We show that guide RNA-target strand DNA hybridization conformationally activates Cas12a, triggering its trans-acting, non-specific, single-stranded DNase activity. In turn, cis cleavage of double-stranded DNA targets is a result of protospacer adjacent motif (PAM)-dependent DNA duplex unwinding, electrostatic stabilization of the displaced non-target DNA strand, and ordered sequential cleavage of the non-target and target DNA strands. Cas12a releases the PAM-distal DNA cleavage product and remains bound to the PAM-proximal DNA cleavage product in a catalytically competent, trans-active state. Together, these results provide a revised model for the molecular mechanisms of both the cis- and the trans-acting DNase activities of Cas12a enzymes, enabling their further exploitation as genome editing tools.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

An Extracellular, Ca2+-Activated Nuclease (EcnA) Mediates Transformation in a Naturally Competent Archaeon.

Transformation, the uptake of DNA directly from the environment, is a major driver of gene flow in microbial populations. In bacteria, DNA uptake requires a nuclease that processes dsDNA to ssDNA, which is subsequently transferred into the cell and incorporated into the genome. However, the process of DNA uptake in archaea is still unknown. Previously, we cataloged genes essential to natural transformation in Methanococcus maripaludis, but few homologs of bacterial transformation-associated genes were identified. Here, we characterize one gene, MMJJ_16440 (named here as ecnA), to be an extracellular nuclease. We show that EcnA is Ca2+-activated, present on the cell surface, and essential for transformation. While EcnA can degrade several forms of DNA, the highest activity was observed with ssDNA as a substrate. Activity was also observed with circular dsDNA, suggesting that EcnA is an endonuclease. This is the first biochemical characterization of a transformation-associated protein in a member of the archaeal domain and suggests that both archaeal and bacterial transformation initiate in an analogous fashion.

Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a.

The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing.

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.

Origin of iodine preferential attack at sulfur in phosphorothioate and subsequent P-O or P-S bond dissociation.

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.

Structures, Mechanisms, and Functions of His-Me Finger Nucleases.

His-Me finger (also called HNH or ßßα-me) nucleases, are a large superfamily of nucleases that share limited sequence homology, but all members carry a highly similar catalytic motif exhibiting a ßßα topology. This review represents a structural comparison of His-Me finger nucleases, summarizing their substrate-binding and recognition strategies, mechanisms of enzymatic hydrolysis, cellular functions, and the various means of activity regulation. His-Me finger nucleases usually function as monomers, making a single nick in nucleic acids to degrade foreign or host genomes, or as homodimers that introduce double-stranded DNA breaks for DNA restriction, integration, recombination, and repair. Various cellular neutralizing machineries have evolved to regulate the activity of His-Me finger nucleases, thereby maintaining genome integrity and cellular functionality.

Characterization and biological prospects of various calcined temperature-V2O5 nanoparticles synthesized by Citrus hystrix fruit extract.

In this study, a simple green synthesis of vanadium pentoxide nanoparticles (VNPs) was prepared by the extract of Kaffir lime fruit (Citrus hystrix) as a green reducing and stabilizing agent, along with the investigation of calcination temperature was carried out at 450 and 550 °C. It was affirmed that, at higher temperature (550 °C), the VNPs possessed a high degree crystalline following the construction of (001) lattice diffraction within an increase in crystalline size from 47.12 to 53.51 nm, although the band gap of the materials at 450 °C was lower than that of the VNPs-550 (2.53 versus 2.66 eV, respectively). Besides, the materials were assessed for the potential bioactivities toward antibacterial, antifungal, DNA cleavage, anti-inflammatory, and hemolytic performances. As a result, the antibacterial activity, with minimal inhalation concentration (MIC) < 6.25 µg/mL for both strains, and fungicidal one of the materials depicted the dose-dependent effects. Once, both VNPs exhibited the noticeable efficacy of the DNA microbial damage, meanwhile, the outstanding anti-inflammatory agent was involved with the IC50 of 123.636 and 227.706 µg/mL, accounting for VNPs-450 and VNPs-550, respectively. Furthermore, this study also demonstrated the hemolytic potential of the VNPs materials. These consequences declare the prospects of the VNPs as the smart and alternative material from the green procedure in biomedicine.

Enzyme-Programmed Self-Assembly of Nanoparticles.

Nanoparticles are a hot topic in the field of nanomaterial research due to their excellent physical and chemical properties. In recent years, DNA-directed nanoparticle self-assembly technology has been widely applied to the development of numerous complex nanoparticle superstructures. Due to the inherent stability and surface electric repulsion of nanoparticles, it is difficult to make nanoparticle superstructures respond to molecular signals in the external environment. In fact, enzyme-programmed molecular systems are developed to allow diverse functions, including logical operations, signal amplification, and dynamic assembly control. Therefore, combining enzyme-controlled DNA systems may endow nanoparticle assembly systems with more flexibility in program design, allowing them to respond to a variety of external signals. In this review, we summarize the basic principles of enzyme-controlled DNA/nanoparticle self-assembly and introduce its applications in heavy metal detection, gene expression, proteins inside living cells, cancer cell therapy, and drug delivery. With the continuous development of new nanoparticle materials and the increasing functionality of enzyme DNA circuits, enzyme-directed DNA/nanoparticle self-assembled probe technology is expected to see significant future development.

Necessity of HuR/ELAVL1 for the activation-induced cytidine deaminase-dependent decrease in topoisomerase 1 in antibody diversification.

Activation-induced cytidine deaminase (AID)-dependent DNA cleavage is the initial event of antibody gene-diversification processes such as class switch recombination (CSR) and somatic hypermutation (SHM). We previously reported the requirement of an AID-dependent decrease of topoisomerase 1 (Top1) for efficient DNA cleavage, but the underlying molecular mechanism has remained elusive. This study focuses on HuR/ELAVL1, a protein that binds to AU-rich elements in RNA. HuR-knockout (KO) CH12 cells derived from murine B lymphoma cells were found to have lower CSR and hypermutation efficiencies due to decreased AID-dependent DNA cleavage levels. The HuR-KO CH12 cells do not show impairment in cell cycles and Myc expression, which have been reported in HuR-reduced spleen B cells. Furthermore, drugs that scavenge reactive oxygen species (ROS) do not rescue the lower CSR in HuR-KO CH12 cells, meaning that ROS or decreased c-Myc protein amount is not the reason for the deficiencies of CSR and hypermutation in HuR-KO CH12 cells. We show that HuR binds to Top1 mRNA and that complete deletion of HuR abolishes AID-dependent repression of Top1 protein synthesis in CH12 cells. Additionally, reduction of CSR to IgG3 in HuR-KO cells is rescued by knockdown of Top1, indicating that elimination of the AID-dependent Top1 decrease is the cause of the inefficiency of DNA cleavage, CSR and hypermutation in HuR-KO cells. These results show that HuR is required for initiation of antibody diversification and acquired immunity through the regulation of AID-dependent DNA cleavage by repressing Top1 protein synthesis.

An In-Silico Drug Designing Approach Attempted on a Newly Synthesized Co(II) Complex along with its Other Biological Activities: A Combined Investigation of both Experimental and Theoretical Aspects.

A new Co (II) complex incorporating a novel Schiff base ligand acquired from the condensation of 3,3'-Methylenedianiline and 2-Hydroxy-5-bromobenzaldehyde was synthesized and characterized. The synthesized complex was air and moisture stable, monomeric, and non-electrolytic in nature. Based on physical and spectral studies, tetrahedral conformation was ascribed to the synthesized Co (II) complex.Density Functional Theory (DFT) was used to analysis different electronic parameters of the optimized structure of Co(II) complex to reveal its stability.Using different analytic and spectroscopic techniques, the new Co (II) complex was established to interact with DNA quite effectively and works as an efficient metallo intercalators. The synthesized complex was discovered to cleave DNA significantly, so it can be inferred that the complex will inhibit the growth of pathogens. Molecular docking was performed to check the binding affinity of the cobalt complex with different receptors, responsible for different diseases. Proteins like progesterone receptor and induced myeloid leukemia cell differentiation Mcl-1 protein showed high binding affinity with this complex, and hence the complex might have some implications for inhibition of progesterone hormones in biological systems. Biological activity of the Co (II) complex was also predicted through computational analysis with SwissADME.Using strains of Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, and Staphylococcus aureus, an in vitro antibacterial activity of the ligand and Co (II) complex was carried out. This activity was further validated by a molecular docking investigation.

Antibacterial and antioxidant activity of gold and silver nanoparticles in dextran-polyacrylamide copolymers.

Search for new antimicrobial agents is of great significance due to the issue of antimicrobial resistance, which nowadays has become more important than many diseases. The aim of this study was to evaluate the toxicity and biological effects of a dextran-graft-polyacrylamide (D-PAA) polymer-nanocarrier with/without silver or gold nanoparticles (AgNPs/D-PAA and AuNPs/D-PAA, respectively) to analyze their potential to replace or supplement conventional antibiotic therapy. The toxicity of nanocomplexes against eukaryotic cells was assessed on primary dermal fibroblasts using scratch, micronucleus and proliferation assays. DPPH (2,2-diphenyl-1-picrylhydrazylradical) assay was used to evaluate the antioxidant capacity of D-PAA, AgNPs/D-PAA and AuNPs/D-PAA. DNA cleavage, antimicrobial and biofilm inhibition effects of nanocomplexes were investigated. Nanocomplexes were found to be of moderate toxicity against fibroblasts with no genotoxicity observed. AgNPs/D-PAA reduced motility and proliferation at lower concentrations compared with the other studied nanomaterials. AgNPs/D-PAA and AuNPs/D-PAA showed radical scavenging capacities in a dose-dependent manner. The antimicrobial activity of AgNPs/D-PAA against various bacteria was found to be much higher compared to D-PAA and AuNPs/D-PAA, especially against E. hirae, E. faecalis and S. aureus, respectively. D-PAA, AgNPs/D-PAA and AuNPs/D-PAA showed DNA-cleaving and biofilm inhibitory activity, while AgNPs/D-PAA displayed the highest anti-biofilm activity. AgNPs/D-PAA and AuNPs/D-PAA were characterized by good antimicrobial activity. According to the findings of the study, AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of new antimicrobial agents, the fight against biofilms, sterilization and disinfection processes. Our findings confirm the versatility of nanosystems based on dextran-polyacrylamide polymers and indicate that AgNPs/D-PAA and AuNPs/D-PAA can be evaluated as alternatives for the preparation of novel antimicrobial agents.

Mg2+-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage.

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)-distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

Multi-Hydrogen Bonding on Quaternized-Oligourea Receptor Facilitated Its Interaction with Bacterial Cell Membranes and DNA for Broad-Spectrum Bacteria Killing.

Herein, we report a new strategy for the design of antibiotic agents based on the electrostatic interaction and hydrogen bonding, highlighting the significance of hydrogen bonding and the increased recognition sites in facilitating the interaction with bacterial cell membranes and DNA. A series of quaternary ammonium functionalized urea-based anion receptors were studied. While the monodentate mono-urea M1, bisurea M2, and trisurea M3 failed to break through the cell membrane barrier and thus could not kill bacteria, the extended bidentate dimers D1-D3 presented gradually increased membrane penetrating capabilities, DNA conformation perturbation abilities, and broad-spectrum antibacterial activities against E. coli, P. aeruginosa, S. aureus, E. faecalis, and S. epidermidis.

High-Resolution Structure of the Nuclease Domain of the Human Parvovirus B19 Main Replication Protein NS1.

Two new structures of the N-terminal domain of the main replication protein, NS1, of human parvovirus B19 (B19V) are presented here. This domain (NS1-nuc) plays an important role in the "rolling hairpin" replication of the single-stranded B19V DNA genome, recognizing origin of replication sequences in double-stranded DNA, and cleaving (i.e., nicking) single-stranded DNA at a nearby site known as the terminal resolution site (trs). The three-dimensional structure of NS1-nuc is well conserved between the two forms, as well as with a previously solved structure of a sequence variant of the same domain; however, it is shown here at a significantly higher resolution (2.4 Å). Using structures of NS1-nuc homologues bound to single- and double-stranded DNA, models for DNA recognition and nicking by B19V NS1-nuc are presented that predict residues important for DNA cleavage and for sequence-specific recognition at the viral origin of replication. IMPORTANCE The high-resolution structure of the DNA binding and cleavage domain of the main replicative protein, NS1, from the human-pathogenic virus human parvovirus B19 is presented here. Included also are predictions of how the protein recognizes important sequences in the viral DNA which are required for viral replication. These predictions can be used to further investigate the function of this protein, as well as to predict the effects on viral viability due to mutations in the viral protein and viral DNA sequences. Finally, the high-resolution structure facilitates structure-guided drug design efforts to develop antiviral compounds against this important human pathogen.

Evaluation of anti-glioma effects of benzothiazoles as efficient apoptosis inducers and DNA cleaving agents.

Glioma is the fast-growing, aggressive, and prevalent brain cancer with a great level of morbidity and mortality. Current therapy is usually found insufficient for glioma treatment. In the course of our research attempting to identify effective anti-glioma agents, three benzothiazole derivatives (1-3) were examined on U251 glioma cells. Among these derivatives, compound 3 was found to have the strongest cytotoxic effect on glioma cells with an IC50 value of 9.84 ± 0.64 µM in reference to cisplatin (IC50 = 8.41 ± 1.27 µM). Further mechanism of anti-glioma effects of compound 3 was characterized by the determination of its apoptotic effects in glioma cells and DNA cleaving capacity. Compound 3 caused a significant apoptotic death of U251 cell line. Besides, this compound cleaved DNA with FeSO4, H2O2 and ascorbic acid system. Molecular docking results also showed that compound 3 possessed a significant binding potential to DNA via important π-π stacking interaction with DG-16. Some pharmacokinetic determinants of compound 3 complied with standard limits making it as an efficient bioavailable anti-glioma drug candidate for upcoming exploration.