TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases.

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA's presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

Mononuclear copper(II) complexes with polypyridyl ligands: synthesis, characterization, DNA interactions/cleavages and in vitro cytotoxicity towards human cancer cells.

DNA being the necessary element in cell regeneration, controlled cellular apoptosis via DNA binding/cleaving is considered an approach to combat cancer cells. The widely prescribed metallodrug cisplatin has shown interactions with the guanine-N7 center, and a plethora of complexes are continually developed to enhance crosslinking properties as well as covalent and non-covalent interactions. Two pentadentate ligands, L1 (1-(6-(1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine) and L2 (1-(6-(1-methyl-1H-benzo[d]imidazol-2-yl)pyridin-2-yl)-N,N-bis(pyridin-2-ylmethyl)methanamine), were synthesized together with their respective copper(II) complexes [1](ClO4)2 and [2](ClO4)2, which crystallized in a trigonal bipyramidal fashion. Different analytical and spectroscopic methods confirmed their formation, and their redox behaviour was also examined. The interactions of salmon sperm DNA (ss-DNA) with these two complexes were explored using absorbance spectroscopy, and they both exhibited a binding affinity (Kb) of ∼104 M-1. Fluorescence quenching experiments with ethidium bromide (EB)-bound DNA (EB-DNA) were also performed, and Stern-Volmer constant (KSV) values of 6.93 × 103 and 2.34 × 104 M-1 for [1](ClO4)2 and [2](ClO4)2, respectively, were obtained. Furthermore, DNA conformational changes due to the interactions of both complexes were validated via circular dichroism. We also assessed the DNA cleavage property of these complexes, which resulted in the linearization of circular plasmid DNA. This finding was supported by studying the growth of MDA-MB-231 breast cancer cells upon treatment with both Cu(II) complexes; IC50 values of 5.34 ± 1.02 µM and 0.83 ± 0.18 µM were obtained for [1](ClO4)2 and [2](ClO4)2, respectively. This validates their affinity towards DNA, and these insights can be further utilized for non-platinum based economical metallodrug development based on first row transition metals.

Ru(II) Complexes with Absorption in the Photodynamic Therapy Window: 1O2 Sensitization, DNA Binding, and Plasmid DNA Photocleavage.

Two Ru(II) complexes, [Ru(pydppn)(bim)(py)]2+ [2; pydppn = 3-(pyrid-2'-yl)-4,5,9,16-tetraaza-dibenzo[a,c]naphthacene; bim = 2,2'-bisimidazole; py = pyridine] and [Ru(pydppn)(Me4bim)(py)]2+ [3; Me4bim = 2,2'-bis(4,5-dimethylimidazole)], were synthesized and characterized, and their photophysical properties, DNA binding, and photocleavage were evaluated and compared to [Ru(pydppn)(bpy)(py)]2+ (1; bpy = 2,2'-bipyridine). Complexes 2 and 3 exhibit broad 1MLCT (metal-to-ligand charge transfer) transitions with maxima at ∼470 nm and shoulders at ∼525 and ∼600 nm that extend to ∼800 nm. These bands are red-shifted relative to those of 1, attributed to the π-donating ability of the bim and Me4bim ligands. A strong signal at 550 nm is observed in the transient absorption spectra of 1-3, previously assigned as arising from a pydppn-centered 3ππ* state, with lifetimes of ∼19 µs for 1 and 2 and ∼270 ns for 3. A number of methods were used to characterize the mode of binding of 1-3 to DNA, including absorption titrations, thermal denaturation, relative viscosity changes, and circular dichroism, all of which point to the intercalation of the pydpppn ligand between the nucleobases. The photocleavage of plasmid pUC19 DNA was observed upon the irradiation of 1-3 with visible and red light, attributed to the sensitized generation of 1O2 by the complexes. These findings indicate that the bim ligand, together with pydppn, serves to shift the absorption of Ru(II) complexes to the photodynamic therapy window, 600-900 nm, and also extend the excited state lifetimes for the efficient production of cytotoxic singlet oxygen.

Pro-CRISPR PcrIIC1-associated Cas9 system for enhanced bacterial immunity.

The CRISPR system is an adaptive immune system found in prokaryotes that defends host cells against the invasion of foreign DNA1. As part of the ongoing struggle between phages and the bacterial immune system, the CRISPR system has evolved into various types, each with distinct functionalities2. Type II Cas9 is the most extensively studied of these systems and has diverse subtypes. It remains uncertain whether members of this family can evolve additional mechanisms to counter viral invasions3,4. Here we identify 2,062 complete Cas9 loci, predict the structures of their associated proteins and reveal three structural growth trajectories for type II-C Cas9. We found that novel associated genes (NAGs) tended to be present within the loci of larger II-C Cas9s. Further investigation revealed that CbCas9 from Chryseobacterium species contains a novel ß-REC2 domain, and forms a heterotetrameric complex with an NAG-encoded CRISPR-Cas-system-promoting (pro-CRISPR) protein of II-C Cas9 (PcrIIC1). The CbCas9-PcrIIC1 complex exhibits enhanced DNA binding and cleavage activity, broader compatibility for protospacer adjacent motif sequences, increased tolerance for mismatches and improved anti-phage immunity, compared with stand-alone CbCas9. Overall, our work sheds light on the diversity and 'growth evolutionary' trajectories of II-C Cas9 proteins at the structural level, and identifies many NAGs-such as PcrIIC1, which serves as a pro-CRISPR factor to enhance CRISPR-mediated immunity.

Discovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs.

CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.

Discovery and characterization of sgRNA-sequence-independent DNA cleavage from CRISPR/Cas9 in mouse embryos.

The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.

Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

Iridium(III)-based minor groove binding complexes as DNA photocleavage agents.

Transition metal complexes containing the qtpy ligand (2':4,4'':4',4'''-quaterpyridyl) are known to be DNA intercalators or minor groove binders. In this study, new tricationic iridium(III) complexes of qtpy are reported. Both [Ir(bpy)2(qtpy)]3+1 and [Ir(phen)2(qtpy)]3+2 display good water solubility as chloride salts. The complexes possess high-energy excited states, which are quenched in the presence of duplex DNA and even by the mononucleotides guanosine monophosphate and adenosine monophosphate. Further studies reveal that although the complexes bind to quadruplex DNA, they display a preference for duplex structures, which are bound with an order of magnitude higher affinities than their isostructural dicationic RuII-analogues. Detailed molecular dynamics simulations confirm that the complexes are groove binders through the insertion of, predominantly, the qtpy ligand into the minor groove. Photoirradiation of 1 in the presence of plasmid DNA confirms that this class of complexes can function as synthetic photonucleases by cleaving DNA.

Structures and activation mechanism of the Gabija anti-phage system.

Prokaryotes have evolved intricate innate immune systems against phage infection1-7. Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB8. GajA functions as a DNA endonuclease that is inactive in the presence of ATP9. Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg2+. GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation.

Electrochemical DNA Cleavage Sensing for EcoRV Activity and Inhibition with an ERGO Electrode.

An electrochemically reduced graphene oxide (ERGO) electrode-based electrochemical assay was developed for rapid, sensitive, and straightforward analysis of both activity and inhibition of the endonuclease EcoRV. The procedure uses a DNA substrate designed for EcoRV, featuring a double-stranded DNA (dsDNA) region labeled with methylene blue (MB) and a single-stranded DNA (ssDNA) region immobilized on the ERGO surface. The ERGO electrode, immobilized with the DNA substrate, was subsequently exposed to a sample containing EcoRV. Upon enzymatic hydrolysis, the cleaved dsDNA fragments were detached from the ERGO surface, leading to a decrease in the MB concentration near the electrode. This diminished the electron transfer efficiency for MB reduction, resulting in a decreased reduction current. This assay demonstrates excellent specificity and high sensitivity, with a limit of detection (LOD) of 9.5 × 10-3 U mL-1. Importantly, it can also measure EcoRV activity in the presence of aurintricarboxylic acid, a known inhibitor, highlighting its potential for drug discovery and clinical diagnostic applications.

T790M mutation upconversion fluorescence biosensor via mild ATRP strategy and site-specific DNA cleavage of restriction endonuclease.

A unique combination of a specific nucleic acid restriction endonuclease (REase) and atom transfer radical polymerization (ATRP) signal amplification strategy was employed for the detection of T790M mutations prevalent in the adjuvant diagnosis of lung cancer. REase selectively recognizes and cleaves T790M mutation sites on double-stranded DNA formed by hybridization of a capture sequence and a target sequence. At the same time, the ATRP strategy resulted in the massive aggregation of upconverted nanoparticles (UCNPs), which significantly improved the sensitivity of the biosensor. In addition, the UCNPs have excellent optical properties and can eliminate the interference of autofluorescence in the samples, thus further improving the detection sensitivity. The proposed upconversion fluorescent biosensor is characterized by high specificity, high sensitivity, mild reaction conditions, fast response time, and a detection limit as low as 0.14 fM. The performance of the proposed biosensor is comparable to that of clinical PCR methods when applied to clinical samples. This work presents a new perspective for assisted diagnosis in the pre-intervention stage of tumor diagnostics in the early stage of precision oncology treatments.

Hydrolytic endonucleolytic ribozyme (HYER) is programmable for sequence-specific DNA cleavage.

Ribozymes are catalytic RNAs with diverse functions including self-splicing and polymerization. This work aims to discover natural ribozymes that behave as hydrolytic and sequence-specific DNA endonucleases, which could be repurposed as DNA manipulation tools. Focused on bacterial group II-C introns, we found that many systems without intron-encoded protein propagate multiple copies in their resident genomes. These introns, named HYdrolytic Endonucleolytic Ribozymes (HYERs), cleaved RNA, single-stranded DNA, bubbled double-stranded DNA (dsDNA), and plasmids in vitro. HYER1 generated dsDNA breaks in the mammalian genome. Cryo-electron microscopy analysis revealed a homodimer structure for HYER1, where each monomer contains a Mg2+-dependent hydrolysis pocket and captures DNA complementary to the target recognition site (TRS). Rational designs including TRS extension, recruiting sequence insertion, and heterodimerization yielded engineered HYERs showing improved specificity and flexibility for DNA manipulation.

Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions.

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the "metal-soup" scenario, also known as "super-stoichiometric" ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu2+) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

Mapping catalytically engaged TOP2B in neurons reveals the principles of topoisomerase action within the genome.

We trapped catalytically engaged topoisomerase IIß (TOP2B) in covalent DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that TOP2Bcc distribution varies with both nucleosome and compartmental chromosome organization. While TOP2Bccs in gene bodies correlate with their level of transcription, highly expressed genes that lack the usually associated chromatin marks, such as H3K36me3, show reduced TOP2Bccs, suggesting that histone posttranslational modifications regulate TOP2B activity. Promoters with high RNA polymerase II occupancy show elevated TOP2B chromatin immunoprecipitation sequencing signals but low TOP2Bccs, indicating that TOP2B catalytic engagement is curtailed at active promoters. Surprisingly, either poisoning or inhibiting TOP2B increases nascent transcription at most genes and enhancers but reduces transcription within long genes. These effects are independent of transcript length and instead correlate with the presence of intragenic enhancers. Together, these results clarify how cells modulate the catalytic engagement of topoisomerases to affect transcription.

Cu(II) flavonoids as potential photochemotherapeutic agents.

Flavonoids, naturally derived polyphenolic compounds, have received significant attention due to their remarkable biochemical properties that offer substantial health benefits to humans. In this work, a series of six Cu(II) flavonoid complexes of the formulation [Cu(L1)(L2)](ClO4) where L1 is 3-hydroxy flavone (HF1, 1 and 4), 4-fluoro-3-hydroxy flavone (HF2, 2 and 5), and 2,6-difluoro-3-hydroxy flavone (HF3, 3 and 6); L2 is 1,10-phenanthroline (phen, 1-3) and 2-(anthracen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip, 4-6) were successfully synthesized, fully characterized and also evaluated for their in vitro photo-triggered cytotoxicity in cancer cells. The single-crystal X-ray diffraction structure of complex 2 shows square pyramidal geometry around the Cu(II) center. The complexes 1-6 showed quasi-reversible cyclic voltammetric responses for the Cu(II)/Cu(I) couple at ∼-0.230 V with a very large ΔEp value of ∼350-480 mV against the Ag/AgCl reference electrode in DMF-0.1 M tetrabutylammonium perchlorate (TBAP) at a scan rate of 50 mV s-1. The complexes were found to have considerable binding propensity for human serum albumin (HSA) and calf thymus DNA (ct-DNA). The complexes displayed remarkable dose-dependent photocytotoxicity in visible light (400-700 nm) in both A549 (human lung cancer) and MCF-7 (human breast cancer) cell lines while remaining significantly less toxic in darkness. They were found to be much less toxic to HPL1D (immortalized human peripheral lung epithelial) normal cells compared to A549 and MCF-7 cancer cells. Upon exposure to visible light, they generate reactive oxygen species, which are thought to be the main contributors to the death of cancer cells. In the presence of visible light, the complexes predominantly elicit an apoptotic mode of cell death. Complex 6 preferentially localizes in the mitochondria of A549 cells.

Phytochemical analysis of carotenoid profile in Mentha piperita and Artemisia vulgaris: cytotoxicity in tumoral cells and evaluation of plasmid DNA cleavage.

Several medicinal plants have been administered to cancer patients attributed to their anticarcinogenic and chemoprotective properties, in addition to lower toxicity compared to traditional therapies. The aim was to investigate the antioxidant properties and carotenoid composition of aqueous extracts of Mentha piperita or Artemisia vulgaris which were previously found to exert beneficial effects on human health through diet. aqueous extracts exhibited potent antioxidant activity. A diversity of carotenoids was identified in these extracts using HPLC-PDA-MS/MS. Both extracts contained predominantly all-trans-lutein as the main component within this class. In order to investigate antioxidant properties, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) techniques were used. The (3-4,5 dimethylthiazol-2, 5 diphenyl tetrazolium bromide) (MTT) and Crystal Violet assays assessed cellular cytotoxicity. Assessments of presence of reactive species were carried out following exposure of oral squamous cell carcinoma cell line (SCC-4) to various aqueous extracts of M piperita or A vulgaris utilizing dichlorofluorescein diacetate (DCFH-DA) and nitric oxide (NO) assays. Exposure to these extracts induced severe cytotoxic effects, which led to investigation of the biochemical and molecular mechanisms underlying this observed effect. Data demonstrated that both solutions induced oxidative stress and DNA damage, especially at higher concentrations using agarose gel subjected to electrophoresis. It is known that exposure to excess amounts of antioxidants results in a prooxidant effect which is beneficial in cancer therapy. Further, the extracts were found to reduce viability of SCC-4 in culture, indicating that this antitumoral activity may be of therapeutic importance and requires further study.

Crystal structure of a phenoxyl radical complex relevant to the metal site of the galactose oxidase enzyme: A facile one-pot synthesis, evidence for hydrogen atom transfer and DNA cleavage via self-activation.

Copper complexes [Cu(L1H)ClO4] (1) and [Cu(L2)NO3] (2), which are relevant to the metal site of the galactose oxidase enzyme, were synthesized and characterized by different spectroscopic methods. L1H2 and L2H2 [where L1H2 stands for 2,2'-((1E,1'E)(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))diphenol and L2H2 stands for 6,6'-((1E,1'E)-(2,2'-(pyridine-2,6-diyl)bis(2-phenylhydrazin-2-yl-1-ylidene))bis(methanylylidene))bis(2,4-di-tert-butylphenol), H stands for dissociable proton] are pentadentate ligands. These ligands provide pyridyl N, two imine N, and two non-innocent phenoxyl and phenolato O donors, forming complex 1 as a non-radical complex, while complex 2 is a phenoxyl radical complex. The molecular structures of complexes 1 and 2 were authenticated by X-ray crystallography. Benzyl alcohol oxidation was investigated, and the conversion of 9,10-dihydroanthracene to anthracene was examined to scrutinize the H-atom abstraction reaction. Nuclease activity with complexes 1 and 2 was investigated by self-activated plasmid DNA (pBR322) cleavage. Non-innocent properties of the ligand-containing phenolato function were investigated by DFT calculations.

A Photoresponsive Homing Endonuclease for Programmed DNA Cleavage.

Homing endonucleases are used in a wide range of biotechnological applications including gene editing, in gene drive systems, and for the modification of DNA structures, arrays, and prodrugs. However, controlling nuclease activity and sequence specificity remain key challenges when developing new tools. Here a photoresponsive homing endonuclease was engineered for optical control of DNA cleavage by partitioning DNA binding and nuclease domains of the monomeric homing endonuclease I-TevI into independent polypeptide chains. Use of the Aureochrome1a light-oxygen-voltage domain delivered control of dimerization with light. Illumination reduced the concentration needed to achieve 50% cleavage of the homing target site by 6-fold when compared to the dark state, resulting in an up to 9-fold difference in final yields between cleavage products. I-TevI nucleases with and without a native I-TevI zinc finger motif displayed different nuclease activity and sequence preference impacting the promiscuity of the nuclease domain. By harnessing an alternative DNA binding domain, target preference was reprogrammed only when the nuclease lacked the I-TevI zinc finger motif. This work establishes a first-generation photoresponsive platform for spatiotemporal activation of DNA cleavage.