Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.

Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5′-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5′-phosphate or a 5′-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide–target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide–target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.

Hydrogel nanoparticle as an artificial nuclease for selective DNA cleavage

CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12aKD2P) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12aKD2P that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12aWT) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.

The type I-FHNH CRISPR–Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states—precatalytic, NTS-cleaved, and post-catalytic—revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5′-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.

Mitochondrial DNA release into the cytosol is a critical event in innate immune activation, often acting as a damage-associated molecular pattern (DAMP) that triggers inflammasome assembly. Here, we demonstrate that NLRP3 is involved in the release of D-loop mtDNA into the cytosol. We further show that NLRP3 interacts with NLRP10. NLRP10-mediated oxidized DNA cleavage involves a Schiff base intermediate and is inhibited by small molecules known to inhibit glycosylases. These findings support a model where NLRP10 interaction with oxidized DNA may contribute to long-term senescence secretory phenotype and modulate inflammasome activation. Our study highlights a novel mechanism by which NLRP10 can respond to mitochondrial stress signals to influence innate immunity and suggests therapeutic potential for targeting these interactions in inflammatory diseases.

DNAzymes conventionally require dissolved metal ions for catalytic functions. Herein, we report that metal surfaces directly activate a self-cleaving DNAzyme (PL) at solid-liquid interfaces. PL exhibits activities on copper, vanadium and tantalum surfaces, within a minimal reaction system comprising only the metal surface, PL and double-distilled water. This interfacial activation is highly material-specific, showing complete absence of activity on plastics, glass or wood etc. Mechanistic studies reveal that dissolved oxygen could react with metal surfaces to generate superoxide anions, which serve as triggers for DNA-cleavage. The reaction shows modulatable characteristics, with inhibition by ethylenediaminetetraacetic acid, catalase, nitroblue tetrazolium and cytochrome c, versus enhancement by vitamin C, glutathione and catechol. Furthermore, metal surface-mediated activation was also observed in F-8, Ag10c and I-R3 DNAzymes, indicating that this phenomenon is not an isolated occurrence. This work establishes macroscopic metals as DNAzyme's cofactors, extending DNAzyme catalysis beyond conventional homogeneous systems to heterogeneous interfacial environments.

Spo11 initiates meiotic recombination by introducing programmed DNA double-strand breaks. DNA cleavage occurs via a topoisomerase-like mechanism involving hybrid active sites formed at the dimer interface. However, in contrast to its topoisomerase relative (Topo VI), Spo11 does not form a stable dimer, likely to prevent uncontrolled DNA cleavage. Here, we investigated the dimerization ofS. cerevisiaeSpo11 in complex with its partners Ski8, Rec102, and Rec104. We show that the Spo11 complex dimerizes transiently on DNA, forming unstable dimeric complexes with duplex and branched DNA substrates. Guided by AlphaFold modeling of a pre-cleavage complex, we identified mutations that reduce dimerization. Surprisingly, DSB formation is resilient to mutagenesis of the Spo11 dimer interface, implying that additional factors promote dimerizationin vivo. Finally, we found that Rec102 exerts a key DNA-binding function, essential for catalysis, and show that it also participates in dimerization throughtranscontacts with Ski8. Our work provides new insights into the mechanism of Spo11 dimerization and the role of its partners in initiating meiotic recombination.

CRISPR/Cas9 technology has revolutionized the field of molecular biology by offering a precise, efficient, and versatile tool for genome editing. The CRISPR/Cas9 system originated from the adaptive immune systems of bacteria, which enables targeted modification of genetic material through guide RNA (gRNA)-directed cleavage of DNA by the Cas9 endonuclease. In recent years, its application in bacterial genome reconstruction has opened new frontiers in the production of biologically active compounds. This review comprehensively explores how CRISPR/Cas9 has been utilized to reprogram metabolic pathways, activate silent biosynthetic gene clusters, and engineer microbial strains with enhanced biosynthetic potential. Model organisms, especially <i>Escherichia coli</i>, <i>Bacillus subtilis</i>, <i>Streptomyces</i>, and <i>Cyanobacteria</i>, have been widely edited to improve the yield and efficiency of antibiotics, industrial enzymes, alkaloids, flavonoids, and biodegradable polymers. We highlight advanced systems involving CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), and multiplex genome editing that allow for refined control of gene expression and metabolic flux. Despite its advantages, challenges remain, including off-target effects, variable transformation efficiency across species, and delivery system limitations. However, recent advances involving Cas12 and Cas13 enzymes, machine learning-aided gRNA design, and synthetic biology integration offer promising strategies to overcome these barriers. The combination of CRISPR tools with computational modelling and bioprocess optimization supports bacteria to be redesigned as “microfactories” for large-scale and sustainable bioproduction. This review not only summarizes current applications but also provides insight into future directions of CRISPR/Cas9 technology in microbial biotechnology, emphasizing its central role in next-generation bioindustrial innovation.

Cytotoxic evaluation of dinuclear ruthenium p-cymene complex with the mononuclear counterpart: A structural perspective.

GIY-YIG homing endonucleases are mobile genetic elements found in phage, bacterial, and organellar genomes. Their modular architecture and sequence-tolerant DNA cleavage properties likely represent evolutionary adaptations to tolerate genetic drift at target sites and enable target-site switching. To investigate modular determinants of GIY-YIG nuclease cleavage preference, we constructed 128 chimeric nucleases by shuffling structural blocks between the prototypical GIY-YIG homing endonuclease I-TevI (CNNNG motif preference) and its isoschizomer I-BmoI (NNNNG preference). Chimeras containing a swapped alpha-helix1 and adjacent loop exhibited altered motif preferences, highlighting this region as a modular determinant, whereas swaps in other regions disrupted activity without altering cleavage preference. Directed evolution of nonconserved residues within this region identified a cluster (R30, K33, E36, C39) where substitutions enabled cleavage of targets poorly recognized by wild-type I-TevI, including variants with reprogrammed preference toward TNNNG and GNNNG motifs. Our findings define a modular and structural basis for DNA cleavage preference in the GIY-YIG nuclease domain and suggest that recombination of structural subunits could accelerate adaptation to new target sites over evolutionary time scales. These findings further support a strategy for engineering GIY-YIG nuclease domains with expanded cleavage motif selectivity.

Four novel mononuclear Cu(II) mixed-ligand complexes [Cu(L)(NN')](ClO4)21-4, where L is bis-((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)amine and NN' is 2,2'-bipyridine (bpy) (1), 1,10-phenanthroline (phen) (2), 2,9-dimethyl-1,10-phenanthroline (2,9-dmp) (3), and dipyrido[3,2-d:2',3'-f]-quinoxaline (dpq) (4), have been synthesized and characterized using different analytical and spectral techniques. The geometry around Cu(II) is distorted square pyramidal for 3, while distorted octahedral for the others. By using salmon sperm DNA (ss-DNA), the DNA-binding affinity of the complexes was investigated. The binding constant (Kb) values vary depending on the NN' co-ligand, 4 > 2 > 1 > 3. Additionally, ethidium bromide (EB)-bound DNA was used in the fluorescence quenching experiments. Interestingly, the order of Stern-Volmer constant (KSV) values is 4 > 2 > 3 > 1, indicating that 4 has the strongest interaction with ss-DNA compared to the other complexes. Using an electrophoresis-based in vitro assay, DNA cleavage was evaluated. The cytotoxicity of complexes 1-4 was evaluated in the human breast cancer cell line MCF7 and the triple-negative breast cancer cell line MDA-MB-231. Complexes 2-4 exhibited IC50 values in the low micromolar concentration range. Although DNA cleavage followed the order 4 > 2 > 3 > 1, the highest cytotoxicity, ROS generation and apoptosis were exhibited by 3 (3 > 2 > 4 > 1), suggesting that ROS-mediated pathways could play a dominant role in determining the anticancer activity of Cu(II) complexes.

Sequence-specific cleavage of DNA under ultrasound is due to intramolecular dynamics.

In-vitro anticancer, DNA cleavage, theoretical studies, and Molecular Docking Studies of Mn(II), Co(II), Ni(II), Cu(II), and Zn(II) complexes with a Novel Schiff Base

In this study, a peripherally tetra-substituted zinc phthalocyanine with 2',3',5',6'-tetrafluoro-4'-carboxyethylthio-benzyloxy groups (2) was synthesized. FT-IR, NMR, MALDI-TOF MS, and UV-Vis spectroscopy techniques were used to characterize all the synthesized compounds. The photochemical and photophysical properties of compound 2 were also examined. The singlet oxygen quantum yield was calculated as 0.49 for compound 2 using only light irradiation in the photochemical method. Additionally, compound 2 showed moderate photostability under intense light irradiation. The biological properties of compound 2, including antioxidant, antidiabetic, antimicrobial, DNA cleavage, and anti-biofilm activities, were evaluated. The antioxidant activity was measured using the DPPH radical scavenging assay, where compound 2 demonstrated 18.12% activity. The antidiabetic potential was assessed through amylase inhibition tests, indicating that compound 2 has potential antidiabetic activity. The antimicrobial activity of compound 2 was tested with the microdilution method. It showed significant activity, especially against Gram-positive and Gram-negative bacteria. Furthermore, the antimicrobial effects of compound 2via photodynamic therapy showed enhanced activity. E. coli was used to evaluate the inhibitory effect of compound 2 on cell viability, demonstrating 100% inhibition. The compound's ability to inhibit biofilm formation of S. aureus and P. aeruginosa was also assessed, with compound 2 showing high biofilm inhibition. The anti-biofilm effect was generally more pronounced against S. aureus than P. aeruginosa. Importantly, the biocompatibility of compound 2 was confirmed in L929 fibroblast cells. While it exhibited concentration-dependent cytotoxicity in the dark, under light irradiation, cell viability remained close to or above the thresholds defined by ISO 10993-5 for non-cytotoxicity. As a result, compound 2 is a multifunctional phthalocyanine derivative that combines diverse biological activities with a safety profile, supporting its potential as a promising candidate for biomedical applications.

Traditional luminol-based electrochemiluminescence (ECL) systems rely on hydrogen peroxide as a co-reactant, but its limited solubility restricts luminescence efficiency and detection accuracy. Herein, a core-shell spherical zinc-based metal-organic framework (Zn-MOF) leverages its metal-to-MOF charge transfer (MMCT) properties to autonomously generate reactive oxygen species (ROS). The zinc core serves as an electron reservoir, facilitating electron injection into the MOF shell via MMCT, enabling ROS generation without exogenous oxidants. This mechanism enhances the ECL signal of luminol-derived carbon dots (L-CDs) through ROS-mediated pathways. Integrating L-CDs with Zn-MOF creates a unique reaction environment that shortens the distance between L-CDs and ROS and stabilizes the L-CDs intermediate active species, thereby improving ECL signal strength and stability in a neutral environment. The system also incorporates the specific binding of ochratoxin A to its aptamer, releasing activated DNA to trigger CRISPR/Cas12a-mediated cleavage of single-stranded DNA (ssDNA) anchored to magnetic beads and dopamine (DA). After magnetic separation, DA-ssDNA is modified on the electrode surface to suppress the initial ECL response. This sensing platform offers a robust solution for detecting mycotoxins in complex matrices, with applications in food safety and environmental monitoring.

The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.

A simple electrochemical platform based on functional nucleic acids mediated DNA walker for detecting Pb2+ and malathion in environmental water and soil samples.

Explorative study of a new Schiff base and its Co2+, Mn2+ and Fe3+ complexes: synthesis, characterization, antimicrobial, anticancer, DNA cleavage activities and DFT calculations

Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells.