The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Richard Chahwan - One of the best experts on this subject based on the ideXlab platform.
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a universal fluorescence based toolkit for real time quantification of dna and rna nuclease activity
Scientific Reports, 2019Co-Authors: Emily C Sheppard, Sally L Rogers, Nicholas J Harmer, Richard ChahwanAbstract:DNA and RNA Nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many Nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and co-factors can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we present a quick, safe, cost-effective, and real-time versatile nuclease assay, which uniquely studies nuclease enzyme kinetics. In conjunction with a substrate library we can now analyse nuclease catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended our analysis to study the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, RNA, and RNA:DNA Nucleases. These Nucleases are involved in DNA repair, immune regulation, and have been associated with various diseases, including cancer and immune disorders.
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a universal fluorescence based toolkit for real time quantification of dna and rna nuclease activity
bioRxiv, 2019Co-Authors: Emily C Sheppard, Sally L Rogers, Nicholas J Harmer, Richard ChahwanAbstract:DNA and RNA Nucleases play a critical role in a growing number of cellular processes ranging from DNA repair to immune surveillance. Nevertheless, many Nucleases have unknown or poorly characterized activities. Elucidating nuclease substrate specificities and regulatory components can support a more definitive understanding of cellular mechanisms in physiology and disease. Using fluorescence-based methods, we have developed a quick, safe, reproducible, cost-effective, and real-time nuclease assay toolkit that could be used for small- and large- scale experimental assays. Additionally, these data can be analysed to determine each reaction's unique enzyme kinetics. We have designed a library of substrates that can be used to study catalytic rates, directionality, and substrate preferences. The assay is sensitive enough to detect kinetics of repair enzymes when confronted with DNA mismatches or DNA methylation sites. We have also extended this assay to consider analysing the kinetics of human single-strand DNA nuclease TREX2, DNA polymerases, and RNA:DNA Nucleases, which are also involved in DNA repair and immune regulation, and have been associated with various disease conditions, including cancer and immune disorders.
Toni Cathomen - One of the best experts on this subject based on the ideXlab platform.
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a novel tale nuclease scaffold enables high genome editing activity in combination with low toxicity
Nucleic Acids Research, 2011Co-Authors: Claudio Mussolino, Robert Morbitzer, Thomas Lahaye, Fabienne Lutge, Nadine Dannemann, Toni CathomenAbstract:Sequence-specific Nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger Nucleases (ZFNs) and megaNucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE Nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nucleaseassociated cytotoxicities. Moreover, the CCR5specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of Nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.
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Quantification of zinc finger nuclease-associated toxicity.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Tatjana I. Cornu, Toni CathomenAbstract:The recent development of artificial zinc finger Nucleases (ZFNs) for targeted genome editing has opened a broad range of possibilities in biotechnology and gene therapy. The ZFN technology allows a researcher to deliberately choose a target site in a complex genome and create appropriate Nucleases to insert a DNA double-strand break (DSB) at that site. Gene editing frequencies of up to 50% in non-selected human cells attest to the power of this technology. Potential side effects of applying ZFNs include toxicity due to cleavage at off-target sites. This can be brought about by insufficient specificity of DNA binding, hence allowing ZFN activity at similar target sequences within the genome, or by activation of the ZFN nuclease domains before the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.
Malcolm F White - One of the best experts on this subject based on the ideXlab platform.
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fuse to defuse a self limiting ribonuclease ring nuclease fusion for type iii crispr defence
Nucleic Acids Research, 2020Co-Authors: Aleksei Samolygo, Januka S Athukoralage, Shirley Graham, Malcolm F WhiteAbstract:Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector Nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary Nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.
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fuse to defuse a self limiting ribonuclease ring nuclease fusion for type iii crispr defence
bioRxiv, 2020Co-Authors: Aleksei Samolygo, Januka S Athukoralage, Shirley Graham, Malcolm F WhiteAbstract:Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector Nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. To avoid this fate, some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary Nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The activities of the two constituent enzymes in the fusion protein cooperate to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.
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ring Nucleases deactivate type iii crispr riboNucleases by degrading cyclic oligoadenylate
Nature, 2018Co-Authors: Januka S Athukoralage, Shirley Graham, Christophe Rouillon, Sabine Gruschow, Malcolm F WhiteAbstract:The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1–3. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. Cyclic oligoadenylate, in turn, binds to and activates riboNucleases and other factors—via a CRISPR-associated Rossman-fold domain—and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the ‘off-switch’ that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This ‘ring nuclease’ is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring Nucleases adds an important insight to the CRISPR system. In the CRISPR type III system, ‘ring’ Nucleases possess a metal-independent mechanism that cleaves cyclic oligoadenylate ring molecules to switch off the antiviral state in cells.
Daniel F. Voytas - One of the best experts on this subject based on the ideXlab platform.
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non transgenic plant genome editing using purified sequence specific Nucleases
Molecular Plant, 2015Co-Authors: Song Luo, Daniel F. Voytas, Thomas Stoddard, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Andrew Coffman, Adam Retterath, Luc Mathis, Feng ZhangAbstract:Sequence-specific Nucleases, including zinc-finger Nucleases, megaNucleases, TAL effector Nucleases (TALENs), and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these Nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host genome.
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mouse genome engineering using designer Nucleases
Journal of Visualized Experiments, 2014Co-Authors: Mario Hermann, Daniel F. Voytas, Tomas Cermak, Pawel PelczarAbstract:Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer Nucleases such as zinc finger Nucleases (ZFNs), transcription activator-like effector Nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.
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dna replicons for plant genome engineering
The Plant Cell, 2014Co-Authors: Nicholas J Baltes, Tomas Cermak, Javier Gilhumanes, Paul Atkins, Daniel F. VoytasAbstract:Sequence-specific Nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific Nucleases (zinc-finger Nucleases, transcription activator-like effector Nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA. In addition to the nuclease-mediated DNA double-strand breaks, gene targeting was promoted by replication of the repair template and pleiotropic activity of the geminivirus replication initiator proteins. We demonstrate the feasibility of using geminivirus replicons to generate plants with a desired DNA sequence modification. By adopting a general plant transformation method, plantlets with a desired DNA change were regenerated in <6 weeks. These results, in addition to the large host range of geminiviruses, advocate the use of replicons for plant genome engineering.
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Genome Engineering of Crops with Designer Nucleases
The Plant Genome, 2012Co-Authors: Shaun J. Curtin, Daniel F. Voytas, Robert M. StuparAbstract:Recent advances in the fi eld of genome engineering indicate that it will soon be routine to make site-directed modifi cations to the genomes of crop species, including targeted mutations, gene insertions, and gene replacements. This new technology will be used to help elucidate gene function and develop new and valuable traits. Key to enabling site-directed genome modifi cations are sequence-specifi c Nucleases that generate targeted double-stranded DNA breaks in genes of interest. To date, three different sequence-specifi c nuclease systems have been used in crop plants: zinc fi nger Nucleases, transcription activator-like effector Nucleases (TALENs), and LAGLIDADG homing endoNucleases, also termed “megaNucleases.” In this review, we report on the current state of genome engineering in crop plants, comparing the different nuclease and gene delivery systems. We also consider some of the limitations that nucleasemediated crop improvement technologies may encounter.
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targeting dna double strand breaks with tal effector Nucleases
Genetics, 2010Co-Authors: Michelle Christian, Adam J Bogdanove, Feng Zhang, Clarice Schmidt, Erin L Doyle, Aaron W. Hummel, Tomas Cermak, Daniel F. VoytasAbstract:Engineered Nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific Nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
Thomas J. Cradick - One of the best experts on this subject based on the ideXlab platform.
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nuclease target site selection for maximizing on target activity and minimizing off target effects in genome editing
Molecular Therapy, 2016Co-Authors: Thomas J. Cradick, Eli J. FineAbstract:The rapid advancement in targeted genome editing using engineered Nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered Nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.
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an online bioinformatics tool predicts zinc finger and tale nuclease off target cleavage
Nucleic Acids Research, 2014Co-Authors: Eli J. Fine, Thomas J. Cradick, Charles L ZhaoAbstract:Although engineered Nucleases can efficiently cleave intracellular DNA at desired target sites, major concerns remain on potential ‘off-target’ cleavage that may occur throughout the genome. We developed an online tool: predicted report of genome-wide nuclease off-target sites (PROGNOS) that effectively identifies off-target sites. The initial bioinformatics algorithms in PROGNOS were validated by predicting 44 of 65 previously confirmed off-target sites, and by uncovering a new off-target site for the extensively studied zinc finger Nucleases (ZFNs) targeting C-C chemokine receptor type 5. Using PROGNOS, we rapidly interrogated 128 potential off-target sites for newly designed transcription activator-like effector Nucleases containing either Asn-Asn (NN) or Asn-Lys (NK) repeat variable di-residues (RVDs) and 3- and 4-finger ZFNs, and validated 13 bona fide offtarget sites for these Nucleases by DNA sequencing. The PROGNOS algorithms were further refined by incorporating additional features of nuclease–DNA interactions and the newly confirmed off-target sites into the training set, which increased the percentage of bona fide off-target sites found within the top PROGNOS rankings. By identifying potential off-target sites in silico, PROGNOS allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of engineered Nucleases for genome editing applications.
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Identification of Off-Target Cleavage Sites of Zinc Finger Nucleases and TAL Effector Nucleases Using Predictive Models
Methods of Molecular Biology, 2014Co-Authors: Eli J. Fine, Thomas J. CradickAbstract:: Using engineered Nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these Nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.
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high throughput cellular screening of engineered nuclease activity using the single strand annealing assay and luciferase reporter
Methods of Molecular Biology, 2014Co-Authors: Thomas J. Cradick, Christopher J AnticoAbstract:: Engineered Nucleases have been used to generate many model organisms and show great promise for therapeutic genome editing. Current methods to evaluate the activity of these Nucleases can be laborious and often are hampered by readouts with small signals and a significant amount of background noise. We present a simple method that utilizes the established single-strand annealing (SSA) assay coupled with a luciferase assay to generate a high-throughput analysis of nuclease activity. Luciferase reporters provide a higher signal and lower background levels than fluorescent reporters. We engineered a commercially available luciferase plasmid (pGL4.51, Promega) to generate a set of nuclease target plasmids that produce a high signal and activity that correlates well with in vitro data. The SSA luciferase assay can discriminate between Nucleases that give similar signals with other nuclease activity assays. The target plasmid and Nucleases are transfected into cells and are generally cultured for 2 days. Luciferase activity is quantified in the same cell culture plate--streamlining the process from transfection to assay. We have used this robust process to investigate the activity of zinc finger Nucleases (ZFNs) and transcription activated-like effector Nucleases (TALENs).
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high throughput cellular screening of engineered nuclease activity using the single strand annealing assay and luciferase reporter
Methods of Molecular Biology, 2014Co-Authors: Thomas J. Cradick, Christopher J AnticoAbstract:: Engineered Nucleases have been used to generate many model organisms and show great promise for therapeutic genome editing. Current methods to evaluate the activity of these Nucleases can be laborious and often are hampered by readouts with small signals and a significant amount of background noise. We present a simple method that utilizes the established single-strand annealing (SSA) assay coupled with a luciferase assay to generate a high-throughput analysis of nuclease activity. Luciferase reporters provide a higher signal and lower background levels than fluorescent reporters. We engineered a commercially available luciferase plasmid (pGL4.51, Promega) to generate a set of nuclease target plasmids that produce a high signal and activity that correlates well with in vitro data. The SSA luciferase assay can discriminate between Nucleases that give similar signals with other nuclease activity assays. The target plasmid and Nucleases are transfected into cells and are generally cultured for 2 days. Luciferase activity is quantified in the same cell culture plate--streamlining the process from transfection to assay. We have used this robust process to investigate the activity of zinc finger Nucleases (ZFNs) and transcription activated-like effector Nucleases (TALENs).
