-
Molecular Cell May 2015
Topics: Animals; Biomedical Research; Biotechnology; CRISPR-Cas Systems; Cryoelectron Microscopy; Deoxyribonucleases; High-Throughput Nucleotide Sequencing; Humans; RNA Interference
PubMed: 26000838
DOI: 10.1016/j.molcel.2015.05.018 -
The Journal of Experimental Medicine Jun 2023Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function...
Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.
Topics: Animals; Mice; Deoxyribonuclease I; Deoxyribonucleases; DNA; Endodeoxyribonucleases; Extracellular Traps; Mammals; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Biofilms
PubMed: 36928522
DOI: 10.1084/jem.20221086 -
Current Opinion in Microbiology Apr 2018CRISPR-Cas systems are adaptive immune systems that protect their hosts from predation by bacteriophages (phages) and parasitism by other mobile genetic elements (MGEs).... (Review)
Review
CRISPR-Cas systems are adaptive immune systems that protect their hosts from predation by bacteriophages (phages) and parasitism by other mobile genetic elements (MGEs). Given the potent nuclease activity of CRISPR effectors, these enzymes must be carefully regulated to minimize toxicity and maximize anti-phage immunity. While attention has been given to the transcriptional regulation of these systems (reviewed in [1]), less consideration has been given to the crucial post-translational processes that govern enzyme activation and inactivation. Here, we review recent findings that describe how Cas nucleases are controlled in diverse systems to provide a robust anti-viral response while limiting auto-immunity. We also draw comparisons to a distinct bacterial immune system, restriction-modification.
Topics: Bacteria; Bacteriophages; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Deoxyribonucleases; Gene Expression Regulation, Bacterial
PubMed: 29169146
DOI: 10.1016/j.mib.2017.11.005 -
Epigenetics & Chromatin Mar 2021The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs...
BACKGROUND
The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation.
RESULTS
In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming.
CONCLUSIONS
We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.
Topics: Chromatin; Chromosomes; Deoxyribonuclease I; Deoxyribonucleases; Genome; Humans
PubMed: 33743768
DOI: 10.1186/s13072-021-00389-5 -
Database : the Journal of Biological... Sep 2022The rapid advancement of sequencing technology, including next-generation sequencing (NGS), has greatly improved sequencing efficiency and decreased cost. Consequently,...
The rapid advancement of sequencing technology, including next-generation sequencing (NGS), has greatly improved sequencing efficiency and decreased cost. Consequently, huge amounts of genomic, transcriptomic and epigenetic data concerning cotton species have been generated and released. These large-scale data provide immense opportunities for the study of cotton genomic structure and evolution, population genetic diversity and genome-wide mining of excellent genes for important traits. However, the complexity of NGS data also causes distress, as it cannot be utilized easily. Here, we presented the cotton omics data platform COTTONOMICS (http://cotton.zju.edu.cn/), an easily accessible web database that integrates 32.5 TB of omics data including seven assembled genomes, resequencing data from 1180 allotetraploid cotton accessions and RNA-sequencing (RNA-seq), small RNA-sequencing (smRNA-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), DNase hypersensitive sites sequencing (DNase-seq) and Bisulfite sequencing (BS-seq). COTTONOMICS allows users to employ various search scenarios and retrieve information concerning the cotton genomes, genomic variation (Single nucleotide polymorphisms (SNPs) and Insertion and Deletion (InDels)), gene expression, smRNA expression, epigenetic regulation and quantitative trait locus (QTLs). The user-friendly web interface offers a variety of modules for storing, retrieving, analyzing and visualizing cotton multi-omics data to diverse ends, thereby enabling users to decipher cotton population genetics and identify potential novel genes that influence agronomically beneficial traits. Database URL: http://cotton.zju.edu.cn.
Topics: Data Management; Deoxyribonucleases; Epigenesis, Genetic; High-Throughput Nucleotide Sequencing; RNA
PubMed: 36094905
DOI: 10.1093/database/baac080 -
Protein & Cell Dec 2016
Topics: Archaea; Archaeal Proteins; Deoxyribonucleases; Gene Editing
PubMed: 27858349
DOI: 10.1007/s13238-016-0344-8 -
Bioinformatics (Oxford, England) Jun 2023Predicting the regulatory function of non-coding DNA using only the DNA sequence continues to be a major challenge in genomics. With the advent of improved optimization...
MOTIVATION
Predicting the regulatory function of non-coding DNA using only the DNA sequence continues to be a major challenge in genomics. With the advent of improved optimization algorithms, faster GPU speeds, and more intricate machine-learning libraries, hybrid convolutional and recurrent neural network architectures can be constructed and applied to extract crucial information from non-coding DNA.
RESULTS
Using a comparative analysis of the performance of thousands of Deep Learning architectures, we developed ChromDL, a neural network architecture combining bidirectional gated recurrent units, convolutional neural networks, and bidirectional long short-term memory units, which significantly improves upon a range of prediction metrics compared to its predecessors in transcription factor binding site, histone modification, and DNase-I hyper-sensitive site detection. Combined with a secondary model, it can be utilized for accurate classification of gene regulatory elements. The model can also detect weak transcription factor binding as compared to previously developed methods and has the potential to help delineate transcription factor binding motif specificities.
AVAILABILITY AND IMPLEMENTATION
The ChromDL source code can be found at https://github.com/chrishil1/ChromDL.
Topics: Algorithms; Benchmarking; DNA; Deoxyribonuclease I; Transcription Factors
PubMed: 37387183
DOI: 10.1093/bioinformatics/btad217 -
The FEBS Journal Sep 2016Recent advances in gene editing with engineered nucleases have transformed our ability to manipulate the genome from diverse organisms for applications ranging from... (Review)
Review
Recent advances in gene editing with engineered nucleases have transformed our ability to manipulate the genome from diverse organisms for applications ranging from biomedical research to disease treatment. A major complication with these engineered nucleases is the binding of the nuclease to unintended genomic sites that share sequence homology with the on-target site. Cleavage of these off-target sites followed by DNA repair using normal cellular DNA repair mechanisms can cause gene mutation or gross chromosome rearrangement. Identification of nuclease-generated off-target sites is a daunting task due to the size and complexity of the mammalian genome. Five unbiased, genome-wide strategies have been developed to detect the off-target cleavage. Some of these strategies reach the sensitivity near the detection limit of directed deep sequencing and have sufficient precision and resolution to objectively assessing the off-target effect of any engineered nuclease. Significant progress has also been made recently to boost the nuclease targeting specificity by protein engineering to modify the structure of the nuclease and alter the interaction with its genomic target. In several studied cases, the off-target effect generated by the modified nuclease is completely eliminated. These modified nucleases significantly improve the overall fidelity of gene editing. These developments will enable gene editing tools to be applied more broadly and safely in basic research and disease treatment.
Topics: Animals; Binding Sites; DNA Breaks, Double-Stranded; DNA Repair; Deoxyribonucleases; Gene Editing; Humans; Protein Engineering; Substrate Specificity; Transcription Activator-Like Effector Nucleases; Zinc Fingers
PubMed: 27208701
DOI: 10.1111/febs.13760 -
Journal of Molecular Biology Feb 2016Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc... (Review)
Review
Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues such as efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk versus benefit analysis and informed consent.
Topics: Animals; Cell Engineering; Deoxyribonucleases; Gene Targeting; Genetic Engineering; Humans; Mammals; Molecular Medicine; Plants; Recombinant Proteins; Recombination, Genetic; Ribonucleases
PubMed: 26506267
DOI: 10.1016/j.jmb.2015.10.014 -
American Journal of Physiology.... May 2017Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile...
Several recent studies have shown that liver injury is associated with the release of DNA from hepatocytes. This DNA stimulates innate immunity and induces sterile inflammation, exacerbating liver damage. Similar mechanisms have been described for acute renal injury. Deoxyribonuclease degrades cell-free DNA and can potentially prevent some of the induced tissue damage. This study analyzed the effects of thioacetamide-induced hepatorenal injury on plasma DNA in rats. Plasma DNA of both nuclear and mitochondrial origin was higher in thioacetamide-treated animals. Administration of deoxyribonuclease resulted in a mild, nonsignificant decrease in total plasma DNA and plasma DNA of mitochondrial origin but not of nuclear origin. This was accompanied by a decrease in bilirubin, creatinine, and blood urea nitrogen as markers of renal function. In conclusion, the study confirmed the hepatotoxic and nephrotoxic effect of thioacetamide. The associated increase in cell-free DNA seems to be involved in hepatorenal pathogenesis because treatment with deoxyribonuclease resulted in a partial prevention of hepatorenal injury. Further experiments will focus on the effects of long-term treatment with deoxyribonuclease in other clinically more relevant models. Clinical studies should test endogenous deoxyribonuclease activity as a potential risk determinant for kidney or liver failure. Thioacetamide-induced hepatorenal injury resulted in higher plasma cell-free DNA. Deoxyribonuclease decreased average cell-free DNA of mitochondrial origin but not nuclear origin. Deoxyribonuclease partially prevented hepatorenal injury in rats.
Topics: Animals; DNA; Deoxyribonucleases; Hepatorenal Syndrome; Male; Rats; Rats, Wistar; Thioacetamide; Treatment Outcome
PubMed: 28209603
DOI: 10.1152/ajpgi.00446.2016