DNA Sites Research Articles

Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.

The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.

Entropy Driven Binding of 2-(4-Aminophenyl)benzothiazole with DNA: An Experimental and Theoretical Insights

Molecular recognition driven by molecular interactions is an intricate part of drug discovery and targeted drug delivery systems due to the dependence of thetherapeutic properties of drugs on interaction with desired target biomolecules as receptors. 2-(4-Aminophenyl) benzothiazole (APB), a heterocyclic molecule containing a benzothiazole nucleus, is known to induce apoptosis besides inhibiting cancer cell development and has been found effective against different microorganisms. This study highlights the spectroscopic and theoretical investigation of binding interactions of APB and ct-DNA. The UV-vis experiments indicate a binding constant of 9.73 ± 0.4 × 105 M-1. The measured binding constants of 1.0 ± 0.2 × 105, 2.4 ± 0.3 × 105 and 3.5 ± 0.3 × 105 at 298, 303 and 308 K, respectively, from fluorescence experiments indicated a dynamic quenching type. The binding was driven by hydrophobic forces with positive ΔHº (64.8467 ± 2 KJ mol–1) and ΔSº (317.12 ± 2 J mol–1 K–1) values besides spontaneity of the process with negative ΔGº values. The binding of APB molecule in the minor groove of ct-DNA was demonstrated by DNA melting tests, viscosity measurements and site marker displacement using ethidium bromide and Hoechst. Additional studies conducted by KI provided support for minor groove binding and NaCl has no impact on the binding electrostatic interactions, while the CD investigations showed no DNA structural alterations. Molecular docking studies also confirmed the experimental findings placing APB in the ct-DNA minor groove.

Direct observation of subunit rotation during DNA strand exchange by serine recombinases

Serine recombinases are proposed to catalyse site-specific recombination by a unique mechanism called subunit rotation. Cutting and rejoining DNA occurs within an intermediate synaptic complex comprising a recombinase tetramer bound to two DNA sites. After double-strand cleavage at both sites, one half of the complex rotates 180° relative to the other, before re-ligation of the DNA ends. We used single-molecule FRET (smFRET) methods to provide compelling direct physical evidence for subunit rotation by recombinases Tn3 resolvase and Sin. Synaptic complexes containing fluorescently labelled DNA show FRET fluctuations consistent with the subunit rotation model. FRET changes were associated with the rotation steps, on a timescale of 0.4–1.1 s−1\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${{\\mbox{s}}}^{-1}$$\\end{document}, as well as opening and closing of the gap between the scissile phosphates during cleavage and ligation. Multiple rounds of recombination were observed within the ~25 s observation period, including frequent consecutive rotation events in the cleaved-DNA state without evidence of intermediate ligation.

Enhanced design of pCMViR-TSC plasmid vector for sustainably high cargo gene expression in mammalian cells.

The first-generation pCMViR-TSC, implemented through the promoter sandwich rule, yields 10- to 100-fold higher gene expression than the standard plasmid used with the CMV (cytomegalovirus) or CAG promoter. However, the vector's shortcomings limit its utility to transient expression only, as it is not suitable for establishing stable transformants in mammalian cells. To overcome this weakness, we here introduce the improved plasmid vector pSAKA-4B, derived from pCMViR-TSC as a second-generation chromosome-insertable vector. This vector facilitates the linear entry of the expression unit into the TTAA site of DNA universally with transposase assistance. The vector is helpful for the indefinite expression of our target gene. The new vector system is proven here to be efficient in establishing stable transformants with a high likelihood of positive clones that exhibit significantly elevated expression levels of the delivered foreign gene. This system, alongside the first-generation vector, is therefore instrumental for diverse basic research endeavors concerning genes, proteins, cells, and animals, and potentially for clinical applications such as gene therapy.

Novel insights into RNA polymerase II transcription regulation: transcription factors, phase separation, and their roles in cardiovascular diseases.

Transcription factors (TFs) are specialized proteins that bind DNA in a sequence-specific manner and modulate RNA polymerase II (Pol II) in multiple steps of the transcription process. Phase separation is a spontaneous or driven process that can form membrane-less organelles called condensates. By creating different liquid phases at active transcription sites, the formation of transcription condensates can reduce the water content of the condensate and lower the dielectric constant in biological systems, which in turn alters the structure and function of proteins and nucleic acids in the condensate. In RNA Pol II transcription, phase separation formation shortens the time at which TFs bind to target DNA sites and promotes transcriptional bursting. RNA Pol II transcription is engaged in developing several diseases, such as cardiovascular disease, by regulating different TFs and mediating the occurrence of phase separation. This review aims to summarize the advances in the molecular mechanisms of RNA Pol II transcriptional regulation, in particular the effect of TFs and phase separation. The role of RNA Pol II transcriptional regulation in cardiovascular disease will be elucidated, providing potential therapeutic targets for the management and treatment of cardiovascular disease.

Best practices for germline variant and DNA methylation analysis of second- and third-generation sequencing data.

This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.

The Intercalator Ethidium Bromide Generates Covalent Adducts at Apurinic/Apyrimidinic Sites in DNA.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

Sumoylation of thymine DNA glycosylase impairs productive binding to substrate sites in DNA

The base excision repair enzyme thymine DNA glycosylase (TDG) protects against mutations by removing thymine or uracil from guanine mispairs and functions in active DNA demethylation by excising 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Post-translational modification of TDG by SUMO (small ubiquitin-like modifier) reduces its glycosylase activity but the mechanism remains unclear. We investigated this problem using biochemical and biophysical approaches and a TDG construct comprising residues 82-340 (of 410) that includes the SUMOylation site and the motif for non-covalent SUMO binding. Single turnover kinetics experiments were collected at multiple enzyme concentrations ([E]) and the hyperbolic dependence of activity (kobs) on [E] yielded the maximal glycosylase activity (kmax), the enzyme concentration giving half-maximal activity (K0.5), and the catalytic efficiency (kmax/K0.5). Sumoylation of TDG (or TDG82-340) causes large reductions in catalytic efficiency for G·T, G·U, G·fC, and G·caC DNA substrates, due largely to weakened substrate affinity (increased K0.5). 19F NMR experiments show that sumoylation of TDG82-340 reduces productive binding to G·U mispairs and dramatically impairs binding to G·T mispairs. A mutation in the TDG SUMO-interacting motif (SIM), E310Q, shown previously to perturb noncovalent binding of SUMO to unmodified TDG, rescues the glycosylase activity of sumoylated TDG82-340. Similarly, NMR studies show the mutation restores productive binding of sumoylated TDG82-340 to G·U and G·T pairs. Together, the results indicate that intramolecular SUMO-SIM interactions mediate the adverse effect of sumoylation on TDG activity and suggest a model whereby the disruption of SUMO-SIM interactions enables productive binding of sumoylated TDG to substrate sites in DNA.

Identification of Specific Hemoglobin Adduct Patterns in Users of Different Tobacco/nicotine Products by Nontargeted GC-MS/MS Analysis.

Tobacco smoke contains several electrophilic constituents which are capable of forming adducts with nucleophilic sites in DNA and proteins like hemoglobin (Hb) and albumin. New nicotine and tobacco products are discussed as less harmful forms of tobacco use compared to smoking combustible cigarettes (CC) due to reduced exposure to harmful constituents. Hence, the adduct profile in users of various tobacco/nicotine products is expected to differ characteristically. In this article, we present a novel nontargeted screening strategy using GC-MS/MS for Hb adducts based on the analysis of the respective derivatized N-terminal valine adducts after modified Edman degradation. We analyzed blood samples from a clinical study with habitual users of CCs, electronic cigarettes, heated tobacco products (HTPs), oral tobacco, nicotine replacement therapy products and nonusers of any tobacco/nicotine products. Our nontargeted approach revealed significant differences in the Hb adduct profiles of the investigated tobacco/nicotine product user groups. Adduct identification was performed by means of an internal database, retention time estimations based on the theoretical boiling points, as well as in-house synthesized reference compounds. Several chemicals that form adducts with Hb could be identified: methylating and ethylating agents, ethylene oxide, acrylonitrile, acrylamide, glycidamide and 4-hydroxybenzaldehyde. Levels were elevated in smokers compared to all other groups for Hb adducts from methylating agents, ethylene oxide, acrylonitrile, acrylamide and glycidamide. Our approach revealed higher concentrations of Hb adducts formed by ethylation, acrylamide and glycidamide in users of HTPs compared to nonusers. However, concentrations for the latter two were still lower than in smokers. Due to their long half-lives, Hb adducts related to acrylonitrile, acrylamide (glycidamide), and ethylene oxide exposure may be useful for the biochemical verification of subjects̀ compliance in longitudinal and cross-sectional studies with respect to smoking and HTP use/abstinence.

Development of β‐Carboline‐Coumarin Based Hybrids as Potential Cytotoxic and Topoisomerase IIα Inhibitors

AbstractCancer remains a global health concern, prompting extensive efforts to develop novel inhibitors targeting the enzyme topoisomerase IIα as potential anti‐cancer agents. Herein, we strategically combined the pharmacophores from natural (β‐carboline) and (coumarin) sources, diversifying them at two distinct points. The in vitro evaluation of cytotoxic properties were estimated on human cancer cell lines, reveals that among the tested compounds, 13 r (isopropyl‐substituted β‐carboline) exhibited remarkable potency with IC50 value of 4.37 μM in the HCT116 cell line. Its selectivity for cancer cells was validated against normal keratinocyte cells, establishing a favourable selectivity index. Further, SAR studies indicate that no substitution (13 r) at C‐6 and C‐7 of β‐carboline, enhance anticancer activity. Assays confirmed its ability to intercalate with DNA, inhibit topoisomerase IIα, induce apoptosis, and disrupt mitochondrial membrane potential in HCT116 cells. Flow cytometric assays gauged the induction of apoptosis using Annexin‐V/PI dual staining and the disruption of mitochondrial membrane potential through JC staining. Results demonstrated that compound 13 r induced apoptosis and dose‐dependent depolarization of the mitochondrial membrane in HCT116 cells. Molecular docking studies further validated that compound 13 r binds within the active site of DNA when complexed with topo IIα, and this binding was stabilized through interactions with DNA base pairs and amino acid residues.

Human 8-oxoguanine glycosylase OGG1 binds nucleosome at the dsDNA ends and the super-helical locations

The human glycosylase OGG1 extrudes and excises the oxidized DNA base 8-oxoguanine (8-oxoG) to initiate base excision repair and plays important roles in many pathological conditions such as cancer, inflammation, and neurodegenerative diseases. Previous structural studies have used a truncated protein and short linear DNA, so it has been unclear how full-length OGG1 operates on longer DNA or on nucleosomes. Here we report cryo-EM structures of human OGG1 bound to a 35-bp long DNA containing an 8-oxoG within an unmethylated Cp-8-oxoG dinucleotide as well as to a nucleosome with an 8-oxoG at super-helical location (SHL)-5. The 8-oxoG in the linear DNA is flipped out by OGG1, consistent with previous crystallographic findings with a 15-bp DNA. OGG1 preferentially binds near dsDNA ends at the nucleosomal entry/exit sites. Such preference may underlie the enzyme’s function in DNA double-strand break repair. Unexpectedly, we find that OGG1 bends the nucleosomal entry DNA, flips an undamaged guanine, and binds to internal nucleosomal DNA sites such as SHL-5 and SHL+6. We suggest that the DNA base search mechanism by OGG1 may be chromatin context-dependent and that OGG1 may partner with chromatin remodelers to excise 8-oxoG at the nucleosomal internal sites.

Crystal structure of a Clostridioides difficile multiple antibiotic resistance regulator (MarR) CD0473 suggests a potential redox-regulated function

Clostridioides difficile may constitute a small part of normal gut microbiota in humans without causing any symptoms, but an uncontrolled growth common to hospitalized patients can cause Clostridioides difficile infection (CDI) leading to severe colonic symptoms. As the bacteria are attaining resistance to various antibiotics worldwide, CDI is becoming a serious public health problem. Although a family of transcription factors called MarR (Multiple antibiotic resistance Regulator) plays a key role in the bacterial response to various environmental stresses including antibiotics, most of the 14 MarRs predicted to exist in the C. difficile genome lack structural or functional studies. In this respect, X-ray crystal structure of a C. difficile MarR CD0473 with a yet unknown function has been determined using a Hg-soaked crystal. In the structure, two closely located flexible conformations of Hg-bound cysteines suggested a possibility of intra-subunit disulfide bridge formation. By searching the neighboring intergenic regions of CD0473, two pseudo-palindromic DNA sites were found and shown to bind the protein. MarR CD0473 binding stronger to the DNA in an oxidizing condition supported further that it may function as a redox regulated switch likely via its oxidized disulfide formation.

Mechanistic model for epigenetic maintenance by methyl-CpG-binding domain proteins.

DNA methylation is a fundamental element of epigenetic regulation that is governed by the MBD protein superfamily, a group of "readers" that share a highly conserved methyl-CpG-binding domain (MBD) and mediate chromatin remodeler recruitment, transcription regulation, and coordination of DNA and histone modification. Previous work has characterized the binding affinity and sequence selectivity of MBD-containing proteins toward palindromes of 5-methylcytosine (5mC) containing 5mCpG dinucleotides, often referred to as single symmetrically methylated CpG sites. However, little is known about how MBD binding is influenced by the prototypical local clustering of methylated CpG sites and the presence of DNA structural motifs encountered, e.g., during DNA replication and transcription. Here, we use Single-Molecule Kinetics through Equilibrium Poisson Sampling (SiMKEPS) to measure precise binding and dissociation rate constants of the MBD of human protein MBD1 to DNAs with varying patterns of multiple methylated CpG sites and diverse structural motifs. MBD binding is promoted by two major properties of its DNA substrates: 1) tandem (consecutive) symmetrically methylated CpG sites in double-stranded DNA and secondary structures in single-stranded DNA; and 2) DNA forks. Based on our findings, we propose a mechanistic model for how MBD proteins contribute to epigenetic boundary maintenance between transcriptionally silenced and active genome regions.

Genome-wide DNA methylation sequencing reveals the involvement of ferroptosis in hepatotoxicity induced by dietary exposure to food-grade titanium dioxide

BackgroundFollowing the announcement by the European Food Safety Authority that the food additive titanium dioxide (E 171) is unsafe for human consumption, and the subsequent ban by the European Commission, concerns have intensified over the potential risks E 171 poses to human vital organs. The liver is the main organ for food-grade nanoparticle metabolism. It is increasingly being found that epigenetic changes may play an important role in nanomaterial-induced hepatotoxicity. However, the profound effects of E 171 on the liver, especially at the epigenetic level, remain largely unknown.MethodsMice were exposed orally to human-relevant doses of two types of E 171 mixed in diet for 28 and/or 84 days. Conventional toxicology and global DNA methylation analyses were performed to assess E 171-induced hepatotoxicity and epigenetic changes. Whole genome bisulfite sequencing and further ferroptosis protein detection were used to reveal E 171-induced changes in liver methylation profiles and toxic mechanisms.ResultsExposed to E 171 for 28 and/or 84 days resulted in reduced global DNA methylation and hydroxymethylation in the liver of mice. E 171 exposure for 84 days elicited inflammation and damage in the mouse liver, whereas 28-day exposure did not. Whole-genome DNA methylation sequencing disclosed substantial methylation alterations at the CG and non-CG sites of the liver DNA in mice exposed to E 171 for 84 days. Mechanistic analysis of the DNA methylation alterations indicated that ferroptosis contributed to the liver toxicity induced by E 171. E 171-induced DNA methylation changes triggered NCOA4-mediated ferritinophagy, attenuated the protein levels of GPX4, FTH1, and FTL in the liver, and thereby caused ferroptosis.ConclusionsLong-term oral exposure to E 171 triggers hepatotoxicity and induces methylation changes in both CG and non-CG sites of liver DNA. These epigenetic alterations activate ferroptosis in the liver through NCOA4-mediated ferritinophagy, highlighting the role of DNA methylation and ferroptosis in the potential toxicity caused by E 171 in vivo.

Ultrasensitive mass spectrometric quantitation of apurinic/apyrimidinic sites in genomic DNA of mammalian cell lines exposed to genotoxic reagents

The apurinic/apyrimidinic (AP) site is an important intermediate in the DNA base excision repair (BER) pathway, having the potential of being a biomarker for DNA damage. AP sites could lead to the stalling of polymerases, the misincorporation of bases and DNA strand breaks, which might affect physiological function of cells. However, the abundance of AP sites in genomic DNA is very low (less than 2 AP sites/106 nts), which requires a sensitive and accurate method to meet its detection requirements. Here, we described an ultrasensitive quantification method based on a hydrazine-s-triazine reagent (i-Pr2N) labeling for AP sites combining with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The limit of detection reached an ultralow level (40 amol), realizing the most sensitive MS-based quantification for the AP site. To guarantee the accuracy of the quantitative results, the labeling reaction was carried out directly on DNA strands instead of labeling after DNA enzymatic digestion to reduce artifacts that might be produced during the enzymatic process of DNA strands. And selective detection was realized by MS to avoid introducing the false-positive signals from other aldehyde species, which could also react with i-Pr2N. Genomic DNA samples from different mammalian cell lines were successfully analyzed using this method. There were 0.4–0.8 AP sites per 106 nucleotides, and the values would increase 16.1 and 2.75 times when cells were treated with genotoxic substances methyl methanesulfonate and 5-fluorouracil, respectively. This method has good potential in the analysis of a small number of cell samples and clinical samples, is expected to be useful for evaluating the damage level of DNA bases, the genotoxicity of compounds and the drug resistance of cancer cells, and provides a new tool for cell function research and clinical precise treatment.

Investigation of antitumor activity and albumin interaction of new sulfosalicylate-based complex by spectroscopic and computational approaches.

In the present study, the drug delivery by albumin protein and antiproliferetaive activity of new transition metal complex i.e., [Pd (phen)(SSA)] (where phen and SSA represent 1, 10 phenanthroline and sulfosalicylic acid, respectively) was investigated. DFT (density functional theory) calculations were conducted at B3LYP level with 6-311G(d,p)/aug-ccpVTZ-PP basis set for the purpose of geometry optimization, frontier molecular orbital (FMO) analysis, molecular electrostatic potential (MEP), and natural bond orbital (NBO) analysis. Experimental tests were conducted to preliminarily assess the lipophilicity and antitumor activity of the metal complex, resulting in promising findings. In-silico prediction was accomplished to assess its toxicity and bioavailability. To evaluate the binding of the newly formed complex with DNA (which results in halting the cell cycle) or serum albumin protein (drug transporter to the tissues), in-silico molecular modeling was employed. Experimental results (spectroscopic and non-spectroscopic) showed that the new compound interacts with each biomolecule via hydrogen bond and van der Waals interactions. Molecular docking demonstrated the binding of this complex to the DNA groove and site I of BSA occurs mainly through hydrogen bonds. Molecular dynamics simulation confirmed the interactions between [Pd (phen)(SSA)] with DNA or BSA through stable hydrogen bonds.

Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY

Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high mobility-group (HMG) box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY HMG domain bound to a DNA site containing consensus element 5’-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5’-CATTGT/ACAATG-3’, shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever side chain), and Arg20, Asn32 and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles of the distinct box-DNA complexes range from 69-84°, similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36 or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10 or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective, Ala substitutions exhibited significantly decreased transcriptional activation of SRY’s principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.

Engineered transcription-associated Cas9 targeting in eukaryotic cells.

DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.

Creating Meiotic Recombination-Regulating DNA Sites by SpEDIT in Fission Yeast Reveals Inefficiencies, Target-Site Duplications, and Ectopic Insertions.

Recombination hotspot-activating DNA sites (e.g., M26, CCAAT, Oligo-C) and their binding proteins (e.g., Atf1-Pcr1 heterodimer; Php2-Php3-Php5 complex, Rst2, Prdm9) regulate the distribution of Spo11 (Rec12)-initiated meiotic recombination. We sought to create 14 different candidate regulatory DNA sites via bp substitutions in the ade6 gene of Schizosaccharomyces pombe. We used a fission yeast-optimized CRISPR-Cas9 system (SpEDIT) and 196 bp-long dsDNA templates with centrally located bp substitutions designed to ablate the genomic PAM site, create specific 15 bp-long DNA sequences, and introduce a stop codon. After co-transformation with a plasmid that encoded both the guide RNA and Cas9 enzyme, about one-third of colonies had a phenotype diagnostic for DNA sequence changes at ade6. PCR diagnostics and DNA sequencing revealed a diverse collection of alterations at the target locus, including: (A) complete or (B) partial template-directed substitutions; (C) non-homologous end joinings; (D) duplications; (E) bp mutations, and (F) insertions of ectopic DNA. We concluded that SpEDIT can be used successfully to generate a diverse collection of DNA sequence elements within a reporter gene of interest. However, its utility is complicated by low efficiency, incomplete template-directed repair events, and undesired alterations to the target locus.

Nick Induced Dynamics in Supercoiled DNA Facilitates the Protein Target Search Process.

A DNA nick, defined as a discontinuity in a double-stranded DNA molecule where the phosphodiester bond between adjacent nucleotides of one strand is absent due to enzyme action, serves as an effective mechanism to alleviate stress in supercoiled DNA. This stress release is essential for the smooth operation of transcriptional machinery. However, the underlying mechanisms and their impact on protein search dynamics, which are crucial for initiating transcription, remain unclear. Through extensive computer simulations, we unravel the molecular picture, demonstrating that intramolecular stress release due to a DNA nick is driven by a combination of writhing and twisting motions, depending on the nick's position. This stress release is quantitatively manifested as a step-like increase in the linking number. Furthermore, we elucidate that the nicked supercoiled minicircles exhibit enhanced torsional dynamics, promoting rapid conformational changes and frequent shifts in the identities of juxtaposed DNA sites on the plectoneme. The dynamics of the juxtaposition sites facilitates communication between protein and DNA, resulting in faster protein diffusion compared with native DNA with the same topology. Our findings highlight the mechanistic intricacies and underscore the importance of DNA nicks in facilitating transcription elongation by actively managing torsional stress during DNA unwinding by the RNA polymerase.