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Transient gene expression (TGE) in mammalian cells is a well-known approach to the fast expression of recombinant proteins. The human cell line HEK (human embryonic kidney) 293F is widely used in this field, due to its adaptability to grow in suspension to high cell densities in serum-free media, amenability to transfection, and production of recombinant proteins in satisfactory quantities for functional and structural analysis. Amounts of plasmid DNA (pDNA) required in transfections for TGE remain high (usually 1 µg pDNA/mL, or even higher), representing a noticeable proportion of the overall cost. Thus, there is an economic need to reduce amounts of coding pDNA in TGE processes. In this work, amounts of both pDNA and transfecting agent used for TGE in HEK 293F cells have been explored in order to reduce them without compromising (or even improving) the productivity of the process in terms of protein yield. In our hands, minimal polyethyleneimine (PEI) cytotoxicity and optimum protein yields were obtained when transfecting at 0.5 µg pDNA/mL (equal to 0.5 µg pDNA/million cells) and a DNA-to-PEI ratio of 1:3, a trend confirmed for several unrelated recombinant proteins. Thus, carefully tuning pDNA and transfecting agent amounts not only reduces the economic costs but also results in higher recombinant protein yields. These results surely have a direct application and interest for the biopharmaceutical industry, always concerned in increasing productivity while decreasing economic costs. KEY POINTS: • Mammalian cells are widely used to produce recombinant proteins in short times. • Tuning DNA and transfecting agent are of great interest to optimize economic costs. • Reducing DNA and transfecting agent amounts result in higher protein yields., (© 2024. The Author(s).)

Advances in synthetic biology allow the design and manipulation of DNA from the scale of genes to genomes, enabling the engineering of complex genetic information for application in biomanufacturing, biomedicine and other areas. The transfer and subsequent maintenance of large DNA are two core steps in large scale genome rewriting. Compared to small DNA, the high molecular weight and fragility of large DNA make its transfer and maintenance a challenging process. This review outlines the methods currently available for transferring and maintaining large DNA in bacteria, fungi, and mammalian cells. It highlights their mechanisms, capabilities and applications. The transfer methods are categorized into general methods (e.g., electroporation, conjugative transfer, induced cell fusion-mediated transfer, and chemical transformation) and specialized methods (e.g., natural transformation, mating-based transfer, virus-mediated transfection) based on their applicability to recipient cells. The maintenance methods are classified into genomic integration (e.g., CRISPR/Cas-assisted insertion) and episomal maintenance (e.g., artificial chromosomes). Additionally, this review identifies the major technological advantages and disadvantages of each method and discusses the development for large DNA transfer and maintenance technologies., Competing Interests: Declaration of competing interest All the authors declare that they have no competing interests in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Utilization of faeces has long been a popular approach for genetic and ecological studies of wildlife. However, the success of molecular marker genotyping and genome resequencing is often unpredictable due to insufficient enrichment of endogenous DNA in the total faecal DNA that is dominated by bacterial DNA. Here, we report a simple and cheap method named PEERS to predominantly lyse animal cells over bacteria by using sodium dodecyl sulphate so as to discharge endogenous DNA into liquid phase before bacterial DNA. By brief centrifugation, total DNA with enriched endogenous fraction can be extracted from the supernatant using routine methods. Our assessments showed that the endogenous DNA extracted by PEERS was significantly enriched for various types of faeces from different species, preservation time and conditions. It significantly improves the genotyping correctness and efficiency of genome resequencing with the total additional cost of $ 0.1 and a short incubation step to treat a faecal sample. We also provide methods to assess the enrichment efficiency of mitochondrial and nuclear DNA and models to predict the usability of faecal DNA for genotyping of short tandem repeat, single-nucleotide polymorphism and whole-genome resequencing., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Leakage of mitochondrial or nuclear DNA into the cytosol can occur following viral infections, radiation damage, and some cancers. Here, we present an optimized protocol for isolating and quantifying cytosolic DNA from mammalian cells. We describe steps for collecting cytosolic fractions from cells, extracting DNA using columns, and quantifying extracted DNA using qPCR. This straightforward protocol can be completed in as little as 5 hours, and allows for the identification of the source of DNA. For complete details on the use and execution of this protocol, please refer to Jahun et al. 1 ., Competing Interests: Declaration of interests I.G.G. currently works for Exscientia. However, Exscientia had no involvement in the research presented in this paper or the decision to publish the manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA replication through a challenging genomic landscape is coordinated by the replisome, which must adjust to local conditions to provide appropriate replication speed and respond to lesions that hinder its progression. We have previously shown that proteasome shuttle proteins, DNA Damage Inducible 1 and 2 (DDI1/2), regulate Replication Termination Factor 2 (RTF2) levels at stalled replisomes, allowing fork stabilization and restart. Here, we show that during unperturbed replication, RTF2 regulates replisome localization of RNase H2, a heterotrimeric enzyme that removes RNA from RNA-DNA heteroduplexes. RTF2, like RNase H2, is essential for mammalian development and maintains normal replication speed. However, persistent RTF2 and RNase H2 at stalled replication forks prevent efficient replication restart, which is dependent on PRIM1, the primase component of DNA polymerase α-primase. Our data show a fundamental need for RTF2-dependent regulation of replication-coupled ribonucleotide removal and reveal the existence of PRIM1-mediated direct replication restart in mammalian cells., (© 2024. The Author(s).)

Transcription factors contain a DNA-binding domain ensuring specific recognition of DNA target sequences. The family of forkhead (FOX) transcription factors is composed of dozens of paralogs in mammals. The forkhead domain (FHD) is a segment of about 100 amino acids that binds an A-rich DNA sequence. Using DNA and RNA PCR-SELEX, we show that recombinant FOXL2 proteins, either wild-type or carrying the oncogenic variant C134W, recognize similar DNA-binding sites. This suggests that the oncogenic variant does not alter the intrinsic sequence-specificity of FOXL2. Most importantly, we show that FOXL2 binds G2-rich RNA sequences whereas it virtually fails to bind similar sequences in DNA chemistry. Interestingly, a statistically significant subset of genes responding to the knock-down of FOXL2/Foxl2 harbor such G2-rich sequences and are involved in crucial signaling pathways and cellular processes. In addition, we show that FOXA1, FOXO3a and chimeric FOXL2 proteins containing the FHD of the former are also able to interact with some of the preferred FOXL2-binding sequences. Our results point to an unexpected and novel characteristic of the forkhead domain, the biological relevance of which remains to be explored., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Synthetic gene networks in mammalian cells are currently limited to either protein-based transcription factors or RNA-based regulators. Here, we demonstrate a regulatory approach based on circular single-stranded DNA (Css DNA), which can be used as an efficient expression vector with switchable activity, enabling gene regulation in mammalian cells. The Css DNA is transformed into its double-stranded form via DNA replication and used as vectors encoding a variety of different proteins in a wide range of cell lines as well as in mice. The rich repository of DNA nanotechnology allows to use sort single-stranded DNA effectors to fold Css DNA into DNA nanostructures of different complexity, leading the gene expression to programmable inhibition and subsequently re-activation via toehold-mediated strand displacement. The regulatory strategy from Css DNA can thus expand the molecular toolbox for the realization of synthetic regulatory networks with potential applications in genetic diagnosis and gene therapy., (© 2023. Springer Nature Limited.)

Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Electroporation is a non-viral mediated transfection technique, which has the advantages of being harmless, easy to operate, and less expensive. This transfection method can be used for almost all cell types and has gradually become the preferred transfection method for mammalian gene editing. However, further improvements are needed in electroporation efficiency. There is no universal standard electrotransfection step for different types of cells, and the inappropriate electroporation parameters will result in a low transfection efficiency and high cell mortality. Here, we systematically optimized the electrotransfection parameters of piggyBac transposon system into sheep fetal fibroblasts for the first time. We found that the cell transfection efficiency and cell viability could be improved by using traditional cell culture medium DMEM/F12 as an electroporation buffer, and simultaneously using the square-wave pulsing program of 200 V, 2 pulses, 20 ms length, and 20 μg DNA (3 μg/μL) in 4 mm cuvette, and the transfection efficiency and cell viability could eventually reach 78.0% and 40.9%, respectively. The purpose of this study is to provide a method reference and theoretical basis for the plasmid electrotransfection in mammal cells., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Detecting DNA breaks in defined regions of the genome is critical to advancing our understanding of genome stability maintenance. Here, we present exo-FISH, a protocol to label exposed single-stranded DNA in defined repetitive regions of mammalian genomes by combining in vitro restriction enzyme digestion on fixed cells with fluorescence in situ hybridization (FISH). We describe steps for cell harvesting and fixation, slide treatments, and FISH probe hybridization. We then detail procedures for imaging and analysis. For complete details on the use and execution of this protocol, please refer to Saayman et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Epigenetic methylation has been shown to play an important role in transcriptional regulation and disease pathogenesis. Recent advancements in detection techniques have identified DNA N6-methyldeoxyadenosine (6mA) and RNA N6-methyladenosine (m6A) as methylation modifications at the sixth position of adenine in DNA and RNA, respectively. While the distributions and functions of 6mA and m6A have been extensively studied in prokaryotes, their roles in the mammalian brain, where they are enriched, are still not fully understood. In this review, we provide a comprehensive summary of the current research progress on 6mA and m6A, as well as their associated writers, erasers, and readers at both DNA and RNA levels. Specifically, we focus on the potential roles of 6mA and m6A in the fundamental biological pathways of the mammalian genome and highlight the significant regulatory functions of 6mA in neurodegenerative diseases.

The CCCTC-binding factor (CTCF) binds tens of thousands of enhancers and promoters on mammalian chromosomes by means of its 11 tandem zinc finger (ZF) DNA-binding domain. In addition to the 12-15-bp CORE sequence, some of the CTCF binding sites contain 5' upstream and/or 3' downstream motifs. Here, we describe two structures for overlapping portions of human CTCF, respectively, including ZF1-ZF7 and ZF3-ZF11 in complex with DNA that incorporates the CORE sequence together with either 3' downstream or 5' upstream motifs. Like conventional tandem ZF array proteins, ZF1-ZF7 follow the right-handed twist of the DNA, with each finger occupying and recognizing one triplet of three base pairs in the DNA major groove. ZF8 plays a unique role, acting as a spacer across the DNA minor groove and positioning ZF9-ZF11 to make cross-strand contacts with DNA. We ascribe the difference between the two subgroups of ZF1-ZF7 and ZF8-ZF11 to residues at the two positions -6 and -5 within each finger, with small residues for ZF1-ZF7 and bulkier and polar/charged residues for ZF8-ZF11. ZF8 is also uniquely rich in basic amino acids, which allows salt bridges to DNA phosphates in the minor groove. Highly specific arginine-guanine and glutamine-adenine interactions, used to recognize G:C or A:T base pairs at conventional base-interacting positions of ZFs, also apply to the cross-strand interactions adopted by ZF9-ZF11. The differences between ZF1-ZF7 and ZF8-ZF11 can be rationalized structurally and may contribute to recognition of high-affinity CTCF binding sites., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Guidelines identifying appropriate DNA extraction methods for both museum and modern biological samples are scarce or non-existent for mammalian species. Yet, obtaining large-scale genetic material collections are vital for conservation and management purposes. In this study, we evaluated five protocols making use of either spin-column, organic solvents, or magnetic bead-based methods for DNA extraction on skin samples from both modern, traffic-killed (n = 10) and museum (n = 10) samples of European hedgehogs, Ericaneus europaeus. We showed that phenol-chloroform or silica column (NucleoSpin Tissue) protocols yielded the highest amount of DNA with satisfactory purity compared with magnetic bead-based protocols, especially for museum samples. Furthermore, extractions using the silica column protocol appeared to produce longer DNA fragments on average than the other methods tested. Our investigation demonstrates that both commercial extraction kits and phenol-chloroform protocol retrieve acceptable DNA concentrations for downstream processes, from degraded remnants of traffic-killed and museum samples of mammalian specimens. Although all the tested methods could be applied depending on the research questions and laboratory conditions, commercial extraction kits may be preferred due to their effectiveness, safety and the higher quality of the DNA extractions., (© 2023. Springer Nature Limited.)

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi-Goutières syndrome (AGS). Here, we constructed RNase H2 C subunit (RH2C)-knockout mouse fibroblast NIH3T3 cells. Compared with the wild-type NIH3T3 cells, the knockout cells exhibited a decreased single ribonucleotide-hydrolyzing activity and an increased accumulation of ribonucleotides in genomic DNA. Transient expression of wild-type RH2C in the knockout cells increased this activity and decreased this ribonucleotide accumulation. Same events were observed when RH2C variants with an AGS-causing mutation, R69W or K145I, were expressed. These results corresponded with our previous results on the RNase H2 A subunit (RH2A)-knockout NIH3T3 cells and the expression of wild-type RH2A or RH2A variants with an AGS-causing mutation, N213I and R293H, in the RH2A-knockout cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Examination of DNA variation is central to understanding the function of mammalian cells, tissues, and whole bodies. Extraction of high-quality DNA from cells and tissues is necessary for innumerable different experiments. We present protocols for the extraction of DNA from both fresh samples and formalin-fixed tissue. Methods for extracting DNA have been standardized and streamlined over the past couple of decades and many extraction kits are available for a reasonable cost. In addition, many of the extraction procedures can also be automated for even higher throughput sample preparation. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA isolation from whole blood, tissue, and cultured cells Alternate Protocol: DNA extraction using automated machines Basic Protocol 2: DNA isolation from saliva and buccal swabs Basic Protocol 3: DNA isolation from formalin-fixed, paraffin-embedded tissue., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

We investigated gene electrotransfer using electrical short-circuiting via a cell suspension droplet in dielectric oil. An aqueous droplet of a few microliters placed between a pair of electrodes can be deformed by an intense DC electric field depending on the electric field intensity. When a droplet containing suspended cells and plasmid DNA elongates during deformation and connects the electrodes, the resulting short circuit can cause successful gene electrotransfection into various mammalian cells. We also investigated the influence of the electroporation medium on membrane permeabilization and the mechanisms of gene electrotransfection using short-circuiting via an aqueous droplet. One aim of this study was to investigate the influence of the conductivity of electroporation medium on gene electrotransfer stimulated by short-circuiting. It was found that low-conductivity medium with plasmid DNA resulted in a significant decrease in cell viability compared to the high-conductivity medium with plasmid DNA. Therefore, we demonstrated the influence of exogenous DNA on membrane damage stimulated by droplet electroporation using a low-conductivity medium. Thus, electrical stimulation with the combination of plasmid DNA and the low-conductivity medium resulted in tremendous membrane damage. Linearized plasmid DNA stimulated more significant membrane damage than circular DNA. However, the size of linear DNA did not influence the efflux of small intracellular molecules., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tsurusaki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome. In this strategy, we design a pair of in trans prime editing guide RNAs (pegRNAs) for each targeted tandem duplication, which encode the same edits but prime the single-stranded DNA (ssDNA) extension in opposite directions. The reverse transcriptase (RT) template of each extension is designed homologous to the target region of the other single guide RNA (sgRNA) to promote the reannealing of the edited DNA strands and the duplication of the fragment in between. We showed that TD-PE produced robust and precise in situ tandem duplications of genomic fragments ranging from ∼50 bp to ∼10 kb, with a maximal efficiency up to 28.33%. By fine-tuning the pegRNAs, we achieved simultaneous targeted duplication and fragment insertion. Finally, we successfully produced multiple disease-relevant tandem duplications, showing the general utility of TD-PE in genetic research., (© 2023 Jiao et al.; Published by Cold Spring Harbor Laboratory Press.)

In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected-between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration., (© 2023. The Author(s).)

DNA N 6 -methyladenine modification (6mA) is a predominant epigenetic mark in prokaryotes but rarely present in multicellular metazoa. The analytical technologies have been developed for sensitive detection of 6mA, including ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) and single molecule real-time sequencing (SMRTseq). However, it remains challenging to detect 6mA at global level and/or in the context of sequence in multicellular metazoa (including mammals). This mini-review brings insights into current dilemma and potential solutions for the identification and quantifications of 6mA in mammals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

DNA N 6 -methyldeoxyadenosine (6mA) modification was first discovered in Bacterium coli in the 1950s. Over the next several decades, 6mA was recognized as a critical DNA modification in the genomes of prokaryotes and protists. While important in prokaryotes, less is known about the presence and functional roles of DNA 6mA in eukaryotes, particularly in mammals. Taking advantage of recent technology advances that made 6mA detection and sequencing possible, studies over the past several years have brought new insights into 6mA biology in mammals. In this perspective, we present recent progress, discuss challenges, and pose four questions for future research regarding mammalian DNA 6mA., Competing Interests: Declaration of interests C.H. is a scientific founder; a member of the scientific advisory board and equity holder of Aferna Green, Inc. and AccuaDX Inc.; and a scientific co-founder and equity holder of Accent Therapeutics, Inc., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Gene regulation via chemically induced dimerization (CID) is useful for biomedical research. However, the number, type, versatility, and in vivo applications of CID tools remain limited. Here, we demonstrate the development of proteolysis-targeting chimera-based scalable CID (PROTAC-CID) platforms by systematically engineering the available PROTAC systems for inducible gene regulation and gene editing. Further, we show orthogonal PROTAC-CIDs that can fine-tune gene expression at gradient levels or multiplex biological signals with different logic gating operations. Coupling the PROTAC-CID platform with genetic circuits, we achieve digitally inducible expression of DNA recombinases, base- and prime-editors for transient genome manipulation. Finally, we package a compact PROTAC-CID system into adeno-associated viral vectors for inducible and reversible gene activation in vivo. This work provides a versatile molecular toolbox that expands the scope of chemically inducible gene regulation in human cells and mice.

The investigation of complex biological processes requires effective tools for probing the spatiotemporal dynamics of individual cells. Single-cell gene expression analysis, such as RNA in situ hybridization and single-cell PCR, has been demonstrated in various biological applications (Tautz and Pfeifle, Chromosoma 98(2):81-5, 1989; Stahlberg and Bengtsson, Methods 50(4):282-288, 2010; Sanchez-Freire et al., Nat Protoc 7(5):829-838, 2012). However, existing techniques require cell lysis or fixation. The dynamic information and spatiotemporal regulation of the biological process cannot be obtained with these methods. Real-time gene expression analysis in living cells remains an outstanding challenge in the field. Here, we described a single-cell gene expression analysis method in living mammalian cells using a locked nucleic acid/DNA (LNA/DNA) nanobiosensor. This LNA/DNA nanobiosensor consists of a fluorophore-labeled detecting strand and a quenching strand. The fluorophore-labeled LNA probe is designed to hybridize with the target microRNA (miRNA) specifically and displace from the quenching strand, allowing the fluorophore to fluorescence. Large-scale single-cell dynamic gene expression monitoring can be performed using time-lapse microscopy to study spatiotemporal distribution and heterogeneity in gene expression. Multiplex detection of miRNAs can be achieved using different fluorophore-labeled LNA/DNA nanobiosensors. This LNA/DNA protocol is fast, generally applicable, and easily accessible., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recent discoveries have advanced our understanding of recombination initiation beyond the placement of double-stranded DNA breaks (DSBs) from germline replication timing to the dynamic reorganization of chromatin, and defined critical players of recombination initiation. This article focuses on recombination initiation in mammals utilizing the PRDM9 protein to orchestrate crucial stages of meiotic recombination initiation by interacting with the local DNA environment and several protein complexes. The Pioneer Complex with the SNF2-type chromatin remodeling enzyme HELLS, exposes PRDM9-bound DNA. At the same time, a Compass-Complex containing EWSR1, CXXC1, CDYL, EHMT2 and PRDM9 facilitates the association of putative hotspot sites in DNA loops with the chromosomal axis where DSB-promoting complexes are located, and DSBs are catalyzed by the SPO11/TOPOVIBL complex. Finally, homology search is facilitated at PRDM9-directed sites by ANKRD31. The Reader-Writer system consists of PRDM9 writing characteristic histone methylation signatures, which are read by ZCWPW1, promoting efficient homology engagement., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The cell is a fantastic place where molecules dynamically move through the various cellular structures and compartments and meet each other, either transiently or in more stable complexes. These complexes have always a specific biological function; thus, it is important to identify and characterize the interaction between molecules, either DNA/RNA, DNA/DNA, protein/DNA, protein/protein, and so on. polycomb group proteins (PcG proteins) are epigenetic repressors involved in important physiologic processes as development and differentiation. They act on the chromatin through the formation of a repressive environment involving histone modification, recruitment of co-repressors, and chromatin-chromatin interactions. PcG form multiprotein complexes, whose characterization required several approaches. In this chapter, I will describe the co-immunoprecipitation (Co-IP) protocol, an easy method used to identify and analyze multiprotein complexes. In Co-IP, an antibody is used to isolate its target antigen, along with its binding partners, from a mixed sample. The binding partners purified with the immunoprecipitated protein can be identified by Western blot or by mass spectrometry., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library preparation. This technology allows cellular level chromatin accessibility quantification and genomic analysis of the epigenetic information in the chromatin, particularly accessible promoter, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Radiation cancer therapy with ultra-high dose rate (UHDR) exposure, so-called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response to therapy. The aim of this study was to clarify whether a 59.5 MeV proton beam at an UHDR of 48.6 Gy/s could effectively reduce the DNA damage of pBR322 plasmid DNA in solution compared to the conventional dose rate (CONV) of 0.057 Gy/s., Materials and Methods: A simple system, consisting of pBR322 plasmid DNA in 1× Tris-EDTA buffer, was initially employed for proton beam exposure. We then used formamidopyrimidine-DNA glycosylase (Fpg) enzymes. which convert oxidative base damages of oxidized purines to DNA strand breaks, to quantify DNA single strand breaks (SSBs) and double strand breaks (DSBs) by agarose gel electrophoresis., Results: Our findings showed that the SSB induction rate (SSB per plasmid DNA/Gy) at UHDR and the induction of Fpg enzyme sensitive sites (ESS) were significantly reduced in UHDR compared to CONV. However, there was no significant difference in DSB induction and non-DSB cluster damages., Conclusions: UHDR of a 59.5 MeV proton beam could reduce non-clustered, non-DSB damages, such as SSB and sparsely distributed ESS. However, this effect may not be significant in reducing lethal DNA damage that becomes apparent only in acute radiation effects of mammalian cells and in vivo studies.

Regulation of epigenomic functions requires controlled site-specific alteration of epigenetic information. This can be achieved by using designed DNA-binding domains, associated with effector domains, that function as targeted transcription factors or epigenetic modifiers. These effectors have been employed to study the implications of epigenetic modifications, and sequence-specific targeting has been instrumental in understanding the effect of these modification on gene regulation. Ultimately, these tools could be used for therapeutic applications to revert the epigenetic aberrations that have been linked to various diseases. The ability to spatiotemporally control gene expression is especially important for precise regulation of the epigenomic state. In this chapter, we describe the protocol for achieving highly efficient small molecule-inducible transcriptional activation of endogenous mammalian genes, mediated by a dCas9-based system that recruits transcriptional activation domains binding to a chain of concatenated coiled-coil peptides., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA damage plays a critical role in biology and diseases; however, how different types of DNA lesions affect cellular functions is far from clear mostly due to the paucity of high-resolution methods that can map their locations in complex genomes, such as those of mammals. Here, we present the development and validation of SSiNGLe-AP method, which can map a common type of DNA damage, abasic (AP) sites, in a genome-wide and high-resolution manner. We apply this method to six different tissues of mice with different ages and human cancer cell lines. We find a nonrandom distribution of AP sites in the mammalian genome that exhibits dynamic enrichment at specific genomic locations, including single-nucleotide hotspots, and is significantly influenced by gene expression, age and tissue type in particular. Overall, these results suggest that we are only starting to understand the true complexities in the genomic patterns of DNA damage., (© 2022. The Author(s).)

Here we provide a detailed protocol for the enrichment of telomeric repeats from mouse and human cells. The procedure consists of two successive rounds of digestion with frequently cutting restriction enzymes followed by size fractionation. Around 2 mg of genomic DNA is required, and the procedure lasts 5-6 d and yields preparations enriched >800-fold in telomeres. The purified material is suitable for single-molecule analysis of telomere structure, visualizing telomere replication and recombination intermediates by electron microscopy or performing molecular combing at telomeric repeats. No special skills are required for the enrichment procedure, while some assistance is needed in harvesting a large number of plates in a timely fashion at the beginning of the procedure. A smaller-scale version of the protocol that involves one round of digestion and purification requires 200 µg of DNA and enriches telomeres ~50-fold in 4 d is also provided. The latter can be combined with specific labeling for single-molecule analysis of replicating DNA or for long-read sequencing analysis of telomeric repeats. The procedure described here can be adapted to the enrichment of other repetitive elements, based on the use of restriction enzymes that do not cut into the repeat of interest., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

DNA fiber combing is a versatile technique that provides insight into replication fork dynamics at single-molecule resolution. DNA fibers are bound to silanized coverslips and combed, which straightens and aligns the fibers along a single axis. Here, we present a DNA fiber combing protocol that does not use commercial kits; we detail the steps to prepare all materials, reagents, and silanized coverslips. We describe the use of DLD-1 cells, but the protocol is amenable to other cell types., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

DNA mechanical properties play a critical role in every aspect of DNA-dependent biological processes. Recently a high throughput assay named loop-seq has been developed to quantify the intrinsic bendability of a massive number of DNA fragments simultaneously. Using the loop-seq data, we develop a software tool, DNAcycP, based on a deep-learning approach for intrinsic DNA cyclizability prediction. We demonstrate DNAcycP predicts intrinsic DNA cyclizability with high fidelity compared to the experimental data. Using an independent dataset from in vitro selection for enrichment of loopable sequences, we further verified the predicted cyclizability score, termed C-score, can well distinguish DNA fragments with different loopability. We applied DNAcycP to multiple species and compared the C-scores with available high-resolution chemical nucleosome maps. Our analyses showed that both yeast and mouse genomes share a conserved feature of high DNA bendability spanning nucleosome dyads. Additionally, we extended our analysis to transcription factor binding sites and surprisingly found that the cyclizability is substantially elevated at CTCF binding sites in the mouse genome. We further demonstrate this distinct mechanical property is conserved across mammalian species and is inherent to CTCF binding DNA motif., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Genetic modification to restore cell functions in the brain can be performed through the delivery of biomolecules in a minimally invasive manner into live neuronal cells within brain tissues. However, conventional nanoscale needles are too short (lengths of ~10 µm) to target neuronal cells in ~1-mm-thick brain tissues because the neuronal cells are located deep within the tissue. Here, we report the use of nanoscale-tipped wire (NTW) arrays with diameters < 100 nm and wire lengths of ~200 µm to address biomolecule delivery issues. The NTW arrays were manufactured by growth of silicon microwire arrays and nanotip formation. This technique uses pinpoint, multiple-cell DNA injections in deep areas of brain tissues, enabling target cells to be marked by fluorescent protein (FP) expression vectors. This technique has potential for use for electrophysiological recordings and biological transfection into neuronal cells. Herein, simply pressing an NTW array delivers and expresses plasmid DNA in multiple-cultured cells and multiple-neuronal cells within a brain slice with reduced cell damage. Additionally, DNA transfection is demonstrated using brain cells ex vivo and in vivo. Moreover, knockdown of a critical clock gene after injecting a short hairpin RNA (shRNA) and a genome-editing vector demonstrates the potential to genetically alter the function of living brain cells, for example, pacemaker cells of the mammalian circadian rhythms. Overall, our NTW array injection technique enables genetic and functional modification of living cells in deep brain tissue areas, both ex vivo and in vivo., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

The mammalian high mobility group protein AT-hook 2 (HMGA2) houses three motifs that preferentially bind short stretches of AT-rich DNA regions. These DNA binding motifs, known as 'AT-hooks', are traditionally characterized as being unstructured. Upon binding to AT-rich DNA, they form ordered assemblies. It is this disordered-to-ordered transition that has implicated HMGA2 as a protein actively involved in many biological processes, with abnormal HMGA expression linked to a variety of health problems including diabetes, obesity, and oncogenesis. In the current work, the solution binding dynamics of the three 'AT-hook' peptides (ATHPs) with AT-rich DNA hairpin substrates were studied using DNA UV melting studies, fluorescence spectroscopy, native ion mobility spectrometry-mass spectrometry (IMS-MS), solution isothermal titration calorimetry (ITC) and molecular modeling. Results showed that the ATHPs bind to the DNA to form a single, 1:1 and 2:1, 'key-locked' conformational ensemble. The molecular models showed that 1:1 and 2:1 complex formation is driven by the capacity of the ATHPs to bind to the minor and major grooves of the AT-rich DNA oligomers. Complementary solution ITC results confirmed that the 2:1 stoichiometry of ATHP: DNA is originated under native conditions in solution., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Most free-living organisms encode for a deoxyuridine triphosphate nucleotidohydrolase (dUTPase; EC 3.6.1.23). dUTPases represent a family of metalloenzymes that catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, preventing dUTP from being incorporated into DNA by DNA polymerases, maintaining a low dUTP/dTTP pool ratio and providing a necessary precursor for dTTP biosynthesis. Thus, dUTPases are involved in maintaining genomic integrity by preventing the uracilation of DNA. Many DNA-containing viruses, which infect mammals also encode for a dUTPase. This review will summarize studies demonstrating that, in addition to their classical enzymatic activity, some dUTPases possess novel functions that modulate the host innate immune response.

Here we describe TALE.Sense, a versatile platform for sensing DNA sequences in live mammalian cells enabling programmable generation of a customable response that discerns cells containing specified sequence targets. The platform is based on the programmable DNA binding of transcription activator-like effector (TALE) coupled to conditional intein-reconstitution producing a trans-spliced ON-switch for a response circuit. TALE.Sense shows higher efficiency and dynamic range when compared to the reported zinc-finger based DNA-sensor in detecting same DNA sequences. Swapping transcriptional activation modules and introducing SunTag-based amplification loops to TALE.Sense circuits augment detection efficiency of the DNA sensor. The TALE.Sense platform shows versatility when applied to a range of target sites, indicating its suitability for applications to identify live cell variants with anticipated DNA sequences. TALE.Sense could be integrated with other cellular or synthetic circuits by using specified DNA sequences as control-switches, thus expanding the scope in connecting inducible modules for synthetic biology.

N6-methyladenosine (m 6 A) is an RNA modification essential for posttranscriptional regulation of gene expression in eukaryotes. We recently demonstrated that m 6 A decorates the RNA components of R-loops, specific nucleic acid structures consisting of an RNA/DNA hybrid and a single strand of non-template DNA, that represent a major source of genetic instability and, at the same time, contribute to regulation of gene expression in mammalian cells. According to growing body of experimental evidence, adenosine methylation affects stability of these structures and potentially influences various aspects of their metabolism. Here, we present two methods for detection and analysis of m 6 A-containing RNA/DNA hybrids: an immunostaining protocol allowing investigation of their spatial distribution in eukaryotic cells and m 6 A-DNA immunoprecipitation (DIP), an antibody-based technique that permits their genome mapping and locus-specific analysis. In addition to the m 6 A-focused studies, these methodologies can also contribute to elucidating the functional roles of other RNA modifications in R-loop biology., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Advances in "omics" technology have made it possible to study a wide range of cellular phenomena at the single-cell level. Recently, we developed single-cell DNA replication sequencing (scRepli-seq) that measures replication timing (RT) by copy number differences between replicated and unreplicated genomic DNA in replicating single mammalian cells. This method has been used to reveal previously unrecognized static and dynamic natures of several hundred kilobases to a few megabases-scale chromosomal units called RT domains. Because RT domains are highly correlated to A/B compartments detected by Hi-C, scRepli-seq data can be used to predict the 3D organization of the genome in the nuclear space. scRepli-seq, which essentially measures the copy number, can also be applied to study genome instability., (© 2022 S. Karger AG, Basel.)

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The initiation of transcription is a focal point for the regulation of gene activity during mammalian cell differentiation and development. To initiate transcription, RNA polymerase II (Pol II) assembles with general transcription factors into a pre-initiation complex (PIC) that opens promoter DNA. Previous work provided the molecular architecture of the yeast 1-9 and human 10,11 PIC and a topological model for DNA opening by the general transcription factor TFIIH 12-14 . Here we report the high-resolution cryo-electron microscopy structure of PIC comprising human general factors and Sus scrofa domesticus Pol II, which is 99.9% identical to human Pol II. We determine the structures of PIC with closed and opened promoter DNA at 2.5-2.8 Å resolution, and resolve the structure of TFIIH at 2.9-4.0 Å resolution. We capture the TFIIH translocase XPB in the pre- and post-translocation states, and show that XPB induces and propagates a DNA twist to initiate the opening of DNA approximately 30 base pairs downstream of the TATA box. We also provide evidence that DNA opening occurs in two steps and leads to the detachment of TFIIH from the core PIC, which may stop DNA twisting and enable RNA chain initiation.

Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties., Competing Interests: The authors have declared that no competing interests exist.

Programmed DNA recombination in mammalian cells occurs predominantly in a directional manner. While random DNA breaks are typically repaired both by deletion and by inversion at approximately equal proportions, V(D)J and class switch recombination (CSR) of immunoglobulin heavy chain gene overwhelmingly delete intervening sequences to yield productive rearrangement. What factors channel chromatin breaks to deletional CSR in lymphocytes is unknown. Integrating CRISPR knockout and chemical perturbation screening we here identify the Snf2-family helicase-like ERCC6L2 as one such factor. We show that ERCC6L2 promotes double-strand break end-joining and facilitates optimal CSR in mice. At the cellular levels, ERCC6L2 rapidly engages in DNA repair through its C-terminal domains. Mechanistically, ERCC6L2 interacts with other end-joining factors and plays a functionally redundant role with the XLF end-joining factor in V(D)J recombination. Strikingly, ERCC6L2 controls orientation-specific joining of broken ends during CSR, which relies on its helicase activity. Thus, ERCC6L2 facilitates programmed recombination through directional repair of distant breaks.

The genomes of all living cells are under endogenous and exogenous attacks every day, causing diverse genomic lesions. Most of the lesions can be timely repaired by multiple DNA repair pathways. However, some may persist during S-phase, block DNA replication, and challenge genome integrity. Eukaryotic cells have evolved DNA damage tolerance (DDT) to mitigate the lethal effects of arrested DNA replication without prior removal of the offending DNA damage. As one important mode of DDT, translesion DNA synthesis (TLS) utilizes multiple low-fidelity DNA polymerases to incorporate nucleotides opposite DNA lesions to maintain genome integrity. Three different mechanisms have been proposed to regulate the polymerase switching between high-fidelity DNA polymerases in the replicative machinery and one or more specialized enzymes. Additionally, it is known that proliferating cell nuclear antigen (PCNA) mono-ubiquitination is essential for optimal TLS. Given its error-prone property, TLS is closely associated with spontaneous and drug-induced mutations in cells, which can potentially lead to tumorigenesis and chemotherapy resistance. Therefore, TLS process must be tightly modulated to avoid unwanted mutagenesis. In this review, we will focus on polymerase switching and PCNA mono-ubiquitination, the two key events in TLS pathway in mammalian cells, and summarize current understandings of regulation of TLS process at the levels of protein-protein interactions, post-translational modifications as well as transcription and noncoding RNAs. Environ. Mol. Mutagen. 61:680-692, 2020. © 2020 Wiley Periodicals, Inc., (© 2020 Wiley Periodicals, Inc.)

Nucleosomal DNA sequences generally follow a well-known pattern with ∼10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Noninvasive genetic analysis is being used increasingly in field surveys. However, detecting large and middle-sized mammals, such as Carnivora species, using noninvasive samples, such as scat or hair, is time- and labor-intensive due to their low densities and elusive behaviors. As snow tracks are the most frequently encountered natural signs of terrestrial mammals in winter, we employed several methods to recover environmental DNA (eDNA) from snow tracks. We performed both DNA metabarcoding and Sanger sequence analyses, in combination with universal primers on the mitochondrial 12S rRNA gene for mammals and taxon-specific primers on the mitochondrial NADH dehydrogenase subunit 2 gene for Martes species (martens and sables in Mustelidae). Snow samples of four Martes melampus tracks, one Cervus nippon track, one Vulpes vulpes track, and the track of an unidentified Carnivora species were collected from a snowfall area in Kyoto, Japan, in February 2018. Regarding DNA metabarcoding analyses, the sequences of three Carnivora species ( M. melampus, V. vulpes , and Canis lupus familiaris ) and a deer ( C. nippon ) were obtained from their respective snow tracks. Using Sanger sequencing, eDNA on snow tracks was recovered at the species level except for M. melampus using universal primers, while eDNA of M. melampus was sequenced using Martes -specific primers. Snow track surveys in combination with eDNA techniques could dramatically improve the efficiency of monitoring and conservation of mammals., (© 2019 Zoological Society of Japan.)

Living cells communicate information about physiological conditions by producing signaling molecules in a specific timed manner. Different conditions can result in the same total amount of a signaling molecule, differing only in the pattern of the molecular concentration over time. Such temporally coded information can be completely invisible to even state-of-the-art molecular sensors with high chemical specificity that respond only to the total amount of the signaling molecule. Here, we demonstrate design principles for circuits with temporal specificity, that is, molecular circuits that respond to specific temporal patterns in a molecular concentration. We consider pulsatile patterns in a molecular concentration characterized by three fundamental temporal features: time period, duty fraction, and number of pulses. We develop circuits that respond to each one of these features while being insensitive to the others. We demonstrate our design principles using general chemical reaction networks and with explicit simulations of DNA strand displacement reactions. In this way, our work develops building blocks for temporal pattern recognition through molecular computation.

A hereditary system that is based on double-helix DNA sequences provides a stable way to store inherited traits and is favored by most life forms on Earth. However, emerging studies on the phenomenon of non-DNA sequence-based inheritance in multiple species, including mammals, urges a rethinking of the nature of hereditary information carriers and the ways in which they encode heritable traits. In this short opinion piece, we discuss how potential hereditary information carriers such as DNA-associated proteins, epigenetic marks, RNAs and organelles could function via distinct and synergistic modes of action in encoding and transmitting ancestral traits, either inherited or acquired from the environment. In particular, we discuss how the 3D structure of chromatin, in addition to its DNA sequence, may represent a higher-order carrier of hereditary information., Competing Interests: Competing interestsThe author declares no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Here, we report a single-cell DNA replication sequencing method, scRepli-seq, a genome-wide methodology that measures copy number differences between replicated and unreplicated DNA. Using scRepli-seq, we demonstrate that replication-domain organization is conserved among individual mouse embryonic stem cells (mESCs). Differentiated mESCs exhibited distinct profiles, which were also conserved among cells. Haplotype-resolved scRepli-seq revealed similar replication profiles of homologous autosomes, while the inactive X chromosome was clearly replicated later than its active counterpart. However, a small degree of cell-to-cell replication-timing heterogeneity was present, which was smallest at the beginning and the end of S phase. In addition, developmentally regulated domains were found to deviate from others and showed a higher degree of heterogeneity, thus suggesting a link to developmental plasticity. Moreover, allelic expression imbalance was found to strongly associate with replication-timing asynchrony. Our results form a foundation for single-cell-level understanding of DNA replication regulation and provide insights into three-dimensional genome organization.