The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Carlos F. Barbas - One of the best experts on this subject based on the ideXlab platform.
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efficient delivery of Nuclease proteins for genome editing in human stem cells and primary cells
Nature Protocols, 2015Co-Authors: Yifeng Yang, Nan Wang, Sailan Shui, Chidananda Nagamangala Kanchiswamy, Carlos F. BarbasAbstract:Targeted Nucleases, including zinc-finger Nucleases (ZFNs), transcription activator-like (TAL) effector Nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of Nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make Nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating Nuclease protein activity. Once they are constructed, Nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
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zfn talen and crispr cas based methods for genome engineering
Trends in Biotechnology, 2013Co-Authors: Thomas Gaj, Charles A Gersbach, Carlos F. BarbasAbstract:Zinc-finger Nucleases (ZFNs) and transcription activator-like effector Nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric Nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific Nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endoNucleases.
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ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering
Trends in Biotechnology, 2013Co-Authors: Thomas Gaj, Charles A Gersbach, Carlos F. BarbasAbstract:Zinc-finger Nucleases (ZFNs) and transcription activator-like effector Nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric Nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific Nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endoNucleases. © 2013 Elsevier Ltd.
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.
Eli J. Fine - 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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TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
Nucleic Acids Research, 2014Co-Authors: Claudio Mussolino, Jamal Alzubi, Eli J. Fine, Robert Morbitzer, Thomas J. Cradick, Thomas Lahaye, Toni CathomenAbstract:Designer Nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger Nucleases (ZFNs) and RNA-guided endoNucleases has been assessed to some extent, little data are available for transcription activator-like effector-based Nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15–30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer Nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link Nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
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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.
Shengdar Q. Tsai - One of the best experts on this subject based on the ideXlab platform.
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defining crispr cas9 genome wide Nuclease activities with circle seq
Nature Protocols, 2018Co-Authors: Cicera R Lazzarotto, Nhu T Nguyen, Xing Tang, Jose Malagonlopez, Martin J Aryee, Keith J Joung, Shengdar Q. TsaiAbstract:Circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) is a sensitive and unbiased method for defining the genome-wide activity (on-target and off-target) of CRISPR–Cas9 Nucleases by selective sequencing of Nuclease-cleaved genomic DNA (gDNA). Here, we describe a detailed experimental and analytical protocol for CIRCLE-seq. The principle of our method is to generate a library of circularized gDNA with minimized numbers of free ends. Highly purified gDNA circles are treated with CRISPR–Cas9 ribonucleoprotein complexes, and Nuclease-linearized DNA fragments are then ligated to adapters for high-throughput sequencing. The primary advantages of CIRCLE-seq as compared with other in vitro methods for defining genome-wide genome editing activity are (i) high enrichment for sequencing Nuclease-cleaved gDNA/low background, enabling sensitive detection with low sequencing depth requirements; and (ii) the fact that paired-end reads can contain complete information on individual Nuclease cleavage sites, enabling use of CIRCLE-seq in species without high-quality reference genomes. The entire protocol can be completed in 2 weeks, including time for gRNA cloning, sequence verification, in vitro transcription, library preparation, and sequencing.
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Discovering the Genome-Wide Activity of CRISPR-Cas Nucleases
ACS Chemical Biology, 2018Co-Authors: Shengdar Q. TsaiAbstract:Originally discovered as part of an adaptive bacterial defense system against the invasion of foreign phages, programmable CRISPR-Cas Nucleases have emerged as remarkable enzymes with transformative potential for both biological research and clinical application. CRISPR-Cas Nucleases likely evolved in their natural context to tolerate imperfect specificity in order to recognize mutant bacteriophages. However, in the context of biological research and clinical applications, high specificity is generally preferred. For therapeutic applications in particular, it is important to carefully and empirically define the genome-wide activity of engineered Nucleases, as hundreds of millions to billions of cells may be modified in a single therapeutic dose. Over the past several years, a number of both cell-based and in vitro sensitive and unbiased genome-scale methods to define CRISPR-Cas Nuclease specificity have been developed. These methods will play important complementary roles in better understanding their global specificity profiles and identifying optimal Nucleases for applications that demand high precision editing. Improving the sensitivity of mutation detection by next-generation sequencing, developing assays to define the functional consequences of unintended off-target activity Nuclease activity, and understanding the consequences of individual human genetic variation on gene editing activity will be important areas for future research and development. O riginally discovered as part of an adaptive bacterial defense system against the invasion of foreign phages, 1,2 programmable CRISPR-Cas Nucleases have emerged as remarkable enzymes with transformative potential for both biological research and clinical application. Broad and rapid adoption of CRISPR-Cas Nucleases has been catalyzed by the simplicity of the engineered system, which requires only two components: a Nuclease protein and an associated single guide RNA (sgRNA). 3 DNA target recognition requires the presence of a protospacer adjacent motif (PAM) whose sequence specificity is determined by protein:DNA interactions. However, the bulk of the specificity is governed by Watson− Crick base pairing interactions between a sequence specified in the gRNA (protospacer) and a complementary DNA target. For example, the first CRISPR-Cas Nuclease to be widely used, S. pyogenes Cas9, recognizes 20 nt adjacent to an NGG PAM (N 20 NGG). CRISPR-Cas Nucleases likely evolved in their natural context to tolerate imperfect specificity. In an arms race between bacteria and foreign invading bacteriophages, from the bacterial perspective, ideal CRISPR-Cas Nucleases would balance the specificity of DNA target recognition and cleavage to appropriately distinguish between the bacterial and phage genome, but tolerate sufficient imprecision to also recognize mutant phages. This stands in sharp contrast to genome editing applications of CRISPR-Cas Nucleases where recog-nition of sites other than the intended target site is usually undesired. CRISPR-Cas Nuclease specificity is not a simple function of its binding affinity. Unlike zinc finger Nucleases (ZFNs) and transcription activator-like effector Nucleases (TALENs), CRISPR-Cas Nucleases do not passively bind to double-stranded DNA in the major groove but rather actively unwind double-stranded DNA and recognize their targets through a combination of protein:DNA and RNA:DNA interactions. There is a sophisticated proof-reading mechanism for cleavage, mediated by a conformational change that occurs upon recognition of a sufficiently matched RNA:DNA duplex,
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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TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity
Nucleic Acids Research, 2014Co-Authors: Claudio Mussolino, Jamal Alzubi, Eli J. Fine, Robert Morbitzer, Thomas J. Cradick, Thomas Lahaye, Toni CathomenAbstract:Designer Nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger Nucleases (ZFNs) and RNA-guided endoNucleases has been assessed to some extent, little data are available for transcription activator-like effector-based Nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15–30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer Nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link Nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation.
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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).
