24 results on '"Benjamin L. Oakes"'
Search Results
2. Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs
- Author
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Kale Kundert, James E. Lucas, Kyle E. Watters, Christof Fellmann, Andrew H. Ng, Benjamin M. Heineike, Christina M. Fitzsimmons, Benjamin L. Oakes, Jiuxin Qu, Neha Prasad, Oren S. Rosenberg, David F. Savage, Hana El-Samad, Jennifer A. Doudna, and Tanja Kortemme more...
- Subjects
Science - Abstract
Control of CRISPR-Cas9 activity allows for fine-tuning of editing and gene expression. Here the authors use gRNAs modified with RNA aptamers to enable small molecule control in bacterial systems.
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- 2019
- Full Text
- View/download PDF
Catalog
3. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing
- Author
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Benjamin L. Oakes, Danny F. Xia, Elizabeth F. Rowland, Denise J. Xu, Irina Ankoudinova, Jennifer S. Borchardt, Lei Zhang, Patrick Li, Jeffrey C. Miller, Edward J. Rebar, and Marcus B. Noyes
- Subjects
Science - Abstract
Zinc finger nucleases have an established role in genome editing. Here, the authors report a strategy for identifying zinc finger nucleases that discriminate between desired targets and provide genome-wide specificity. more...
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- 2016
- Full Text
- View/download PDF
4. Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
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Rachel J. Lew, Christof Fellmann, Thomas G. Laughlin, Arik Shams, Benjamin L. Oakes, Maria Lukarska, David F. Savage, Jennifer A. Doudna, Brett T. Staahl, Shin Kim, Sean Higgins, and Madeline L. Arnold more...
- Subjects
CRISPR-Cas9 genome editing ,Genetic enhancement ,Science ,General Physics and Astronomy ,Computational biology ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genome editing ,CRISPR-Associated Protein 9 ,Cell Line, Tumor ,DNA-binding proteins ,CRISPR ,Humans ,Protein Interaction Domains and Motifs ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Modularity (networks) ,Multidisciplinary ,Effector ,Cas9 ,Cryoelectron Microscopy ,RNA ,General Chemistry ,DNA ,Single Molecule Imaging ,chemistry ,Protein design ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,RNA, Guide, Kinetoplastida - Abstract
Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics., Proteins evolve through the modular rearrangement of domains. Here the authors introduce MISER, a minimization by iterative size-exclusion and recombination method to make all possible deletions of a protein, uncovering functions for Cas9 domains involved in DNA binding. more...
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- 2021
5. A yeast optogenetic toolkit (yOTK) for gene expression control in Saccharomyces cerevisiae
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Megan N. McClean, Justin Melendez, Michael T Patel, Cameron J. Stewart, Marcus B. Noyes, Jidapas My An-Adirekkun, Stephanie H. Geller, and Benjamin L. Oakes
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Saccharomyces cerevisiae Proteins ,Computer science ,Recombinant Fusion Proteins ,Saccharomyces cerevisiae ,Bioengineering ,Computational biology ,Optogenetics ,Applied Microbiology and Biotechnology ,Article ,03 medical and health sciences ,0302 clinical medicine ,Gene Expression Regulation, Fungal ,Gene expression ,Basic Helix-Loop-Helix Transcription Factors ,Cloning, Molecular ,Promoter Regions, Genetic ,Transcription factor ,030304 developmental biology ,0303 health sciences ,biology ,Arabidopsis Proteins ,030302 biochemistry & molecular biology ,Zinc Fingers ,Promoter ,biology.organism_classification ,Yeast ,Cryptochromes ,030217 neurology & neurosurgery ,Function (biology) ,Biological network ,Transcription Factors ,Biotechnology - Abstract
Optogenetic tools for controlling gene expression are ideal for tuning synthetic biological networks due to the exquisite spatiotemporal control available with light. Here we develop an optogenetic system for gene expression control and integrate it with an existing yeast toolkit allowing for rapid, modular assembly of light-controlled circuits in the important chassis organism Saccharomyces cerevisiae. We reconstitute activity of a split synthetic zinc-finger transcription factor (TF) using light-induced dimerization. We optimize function of this split TF and demonstrate the utility of the toolkit workflow by assembling cassettes expressing the TF activation domain and DNA-binding domain at different levels. Utilizing this TF and a synthetic promoter we demonstrate that light-intensity and duty-cycle can be used to modulate gene expression over the range currently available from natural yeast promoters. This work allows for rapid generation and prototyping of optogenetic circuits to control gene expression in Saccharomyces cerevisiae. more...
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- 2019
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- View/download PDF
6. Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs
- Author
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Benjamin L. Oakes, Tanja Kortemme, Christof Fellmann, James E. Lucas, Kyle E. Watters, Jennifer A. Doudna, Hana El-Samad, Benjamin M. Heineike, Christina M. Fitzsimmons, Andrew H. Ng, David F. Savage, Oren S. Rosenberg, Neha K. Prasad, Kale Kundert, and Jiuxin Qu more...
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0301 basic medicine ,CRISPR-Cas9 genome editing ,Aptamer ,Science ,General Physics and Astronomy ,02 engineering and technology ,Ligands ,Aptamers ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Synthetic biology ,CRISPR-Associated Protein 9 ,MD Multidisciplinary ,Genetics ,CRISPR ,Guide RNA ,lcsh:Science ,Gene ,Subgenomic mRNA ,Gene Editing ,Multidisciplinary ,RNA ,General Chemistry ,DNA ,Aptamers, Nucleotide ,021001 nanoscience & nanotechnology ,Molecular conformation ,Cell biology ,030104 developmental biology ,chemistry ,Generic Health Relevance ,lcsh:Q ,CRISPR-Cas Systems ,0210 nano-technology ,Nucleotide ,Guide ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes., Control of CRISPR-Cas9 activity allows for fine-tuning of editing and gene expression. Here the authors use gRNAs modified with RNA aptamers to enable small molecule control in bacterial systems. more...
- Published
- 2019
- Full Text
- View/download PDF
7. CRISPR-CasX is an RNA-dominated enzyme active for human genome editing
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Eva Nogales, Hannah Spinner, Jonathan Chuck, Basem Al-Shayeb, Natalia Orlova, Enbo Ma, Jun-Jie Liu, Gavin J. Knott, Jennifer A. Doudna, John J Desmarais, Brett T. Staahl, Katherine Baney, Alexander J. Wagner, Julian Brötzmann, Dan Tan, Lucas B. Harrington, Benjamin L. Oakes, and Kian Taylor more...
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0301 basic medicine ,Models, Molecular ,CRISPR-Associated Proteins ,Computational biology ,Biology ,Genome ,Article ,Evolution, Molecular ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Genome editing ,Protein Domains ,Escherichia coli ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Guide RNA ,Gene Silencing ,DNA Cleavage ,Gene Editing ,Multidisciplinary ,Cas9 ,Genome, Human ,Cryoelectron Microscopy ,RNA ,DNA ,030104 developmental biology ,chemistry ,Nucleic acid ,Nucleic Acid Conformation ,Human genome ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,Genome, Bacterial ,RNA, Guide, Kinetoplastida - Abstract
The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a. more...
- Published
- 2019
8. Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules
- Author
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David F. Savage, Benjamin L. Oakes, Rachel J. Lew, Christof Fellmann, Madeline L. Arnold, Maria Lukarska, Brett T. Staahl, Sean Higgins, Jennifer A. Doudna, Arik Shams, and Thomas J. Laughlin
- Subjects
Modularity (networks) ,chemistry.chemical_compound ,Genome editing ,chemistry ,Cas9 ,Effector ,Genetic enhancement ,CRISPR ,RNA ,Computational biology ,Biology ,DNA - Abstract
Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics. more...
- Published
- 2020
- Full Text
- View/download PDF
9. Author Correction: CasX enzymes comprise a distinct family of RNA-guided genome editors
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Jennifer A. Doudna, Julian Brötzmann, Dan Tan, Hannah Spinner, Natalia Orlova, Eva Nogales, Brett T. Staahl, Enbo Ma, Basem Al-Shayeb, Katherine Baney, Alexander J. Wagner, Jun-Jie Liu, Kian Taylor, Benjamin L. Oakes, Gavin J. Knott, Jonathan Chuck, John J Desmarais, and Lucas B. Harrington more...
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Multidisciplinary ,Information retrieval ,Computer science ,Published Erratum ,Section (typography) ,Protein Data Bank (RCSB PDB) ,RNA ,computer.file_format ,Protein Data Bank ,computer ,Genome ,Data availability - Abstract
In this Article, owing to issues with the first 30 nucleotides of the sgRNA, which run in the opposite direction, corrections have been made to the Protein Data Bank (PDB) accessions in the 'Data availability' section, and this also affects Figs. 3, 4, Extended Data Fig. 6, Supplementary Table 1 and Supplementary Video 1. The original Article has been corrected online. See the accompanying Amendment for further details. more...
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- 2019
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10. CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification
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Rayka Yokoo, Jennifer A. Doudna, Benjamin L. Oakes, Adam P. Arkin, Dana C. Nadler, Christof Fellmann, Shawn M. Ren, David F. Savage, Kian Taylor, and Harneet S. Rishi
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Models, Molecular ,ProCas9 ,Cas9-CP ,CRISPR-Associated Proteins ,Computational biology ,Biology ,Genome ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,Article ,Genome engineering ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Models ,Genetics ,CRISPR ,genome editing ,Clustered Regularly Interspaced Short Palindromic Repeats ,CRISPR-Cas ,Biomedicine ,030304 developmental biology ,Gene Editing ,0303 health sciences ,business.industry ,Cas9 ,Effector ,Molecular ,circular permutation ,protein engineering ,DNA ,Circular permutation in proteins ,Biological Sciences ,Infectious Diseases ,chemistry ,RNA ,CRISPR-Cas Systems ,business ,fusion proteins ,030217 neurology & neurosurgery ,Guide ,RNA, Guide, Kinetoplastida ,Biotechnology ,Developmental Biology - Abstract
The ability to engineer natural proteins is pivotal toafuture, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine. more...
- Published
- 2019
11. Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors
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Shannon M. Miller, David F. Savage, Nicole M. Gaudelli, Kevin T. Zhao, Christof Fellmann, David R. Liu, Tony P. Huang, and Benjamin L. Oakes
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Computer science ,Biomedical Engineering ,Bioengineering ,Computational biology ,Applied Microbiology and Biotechnology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Cytosine ,0302 clinical medicine ,CRISPR-Associated Protein 9 ,Humans ,Nucleotide ,030304 developmental biology ,chemistry.chemical_classification ,Gene Editing ,0303 health sciences ,Extramural ,Cas9 ,Nucleotides ,Adenine ,Base (topology) ,Protospacer adjacent motif ,chemistry ,Molecular Medicine ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,Scope (computer science) ,Biotechnology ,Plasmids - Abstract
Base editing requires that the target sequence satisfy the PAM requirement of the Cas9 domain and that the target nucleotide is located within the editing window of the base editor. To increase the targeting scope of base editors, we engineered six optimized adenine base editors (ABEmax variants) that use SpCas9 variants compatible with non-NGG PAMs. To increase the range of target bases that can be modified within the protospacer, we use circularly permuted Cas9 variants to produce four cytosine and four adenine base editors with an editing window expanded from ~4–5 nucleotides to up to ~8–9 nucleotides and reduced byproduct formation. This set of base editors improves the targeting scope of cytosine and adenine base editing., Ed sum: Wider editing windows and different PAM requirements enable a broad set of genomic positions to be targeted with A and C base editors. more...
- Published
- 2018
12. Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs
- Author
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Tanja Kortemme, Christof Fellmann, Benjamin L. Oakes, James E. Lucas, Benjamin M. Heineike, Christina M. Fitzsimmons, Kale Kundert, Jennifer A. Doudna, Andrew H. Ng, David F. Savage, Hana El-Samad, and Kyle E. Watters more...
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0303 health sciences ,Chemistry ,Ligand ,Cell biology ,03 medical and health sciences ,Synthetic biology ,chemistry.chemical_compound ,0302 clinical medicine ,CRISPR ,Guide RNA ,Gene ,030217 neurology & neurosurgery ,DNA ,Function (biology) ,030304 developmental biology ,Subgenomic mRNA - Abstract
The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes. more...
- Published
- 2018
- Full Text
- View/download PDF
13. A systematic survey of the Cys2His2 zinc finger DNA-binding landscape
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Marcus B. Noyes, Denise J. Xu, Benjamin L. Oakes, Joshua L. Wetzel, Anton V. Persikov, Elizabeth F. Rowland, and Mona Singh
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Zinc finger ,Genetics ,Binding Sites ,Systematic survey ,Base pair ,Data Collection ,Context (language use) ,Zinc Fingers ,Computational biology ,DNA ,Biology ,Fusion protein ,Domain (software engineering) ,chemistry.chemical_compound ,chemistry ,Histidine ,Cysteine ,Binding site ,Synthetic Biology and Bioengineering - Abstract
Cys2His2 zinc fingers (C2H2-ZFs) comprise the largest class of metazoan DNA-binding domains. Despite this domain's well-defined DNA-recognition interface, and its successful use in the design of chimeric proteins capable of targeting genomic regions of interest, much remains unknown about its DNA-binding landscape. To help bridge this gap in fundamental knowledge and to provide a resource for design-oriented applications, we screened large synthetic protein libraries to select binding C2H2-ZF domains for each possible three base pair target. The resulting data consist of >160 000 unique domain–DNA interactions and comprise the most comprehensive investigation of C2H2-ZF DNA-binding interactions to date. An integrated analysis of these independent screens yielded DNA-binding profiles for tens of thousands of domains and led to the successful design and prediction of C2H2-ZF DNA-binding specificities. Computational analyses uncovered important aspects of C2H2-ZF domain–DNA interactions, including the roles of within-finger context and domain position on base recognition. We observed the existence of numerous distinct binding strategies for each possible three base pair target and an apparent balance between affinity and specificity of binding. In sum, our comprehensive data help elucidate the complex binding landscape of C2H2-ZF domains and provide a foundation for efforts to determine, predict and engineer their DNA-binding specificities. more...
- Published
- 2015
14. Author Correction: Circularly permuted and PAM-modified Cas9 variants broaden the targeting scope of base editors
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Nicole M. Gaudelli, Tony P. Huang, Kevin T. Zhao, Christof Fellmann, David R. Liu, David F. Savage, Shannon M. Miller, and Benjamin L. Oakes
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Information retrieval ,Scope (project management) ,Computer science ,Published Erratum ,Biomedical Engineering ,Molecular Medicine ,Bioengineering ,Base (topology) ,Applied Microbiology and Biotechnology ,Biotechnology - Published
- 2019
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15. Deep sequencing of large library selections allows computational discovery of diverse sets of zinc fingers that bind common targets
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Elizabeth F. Rowland, Benjamin L. Oakes, Mona Singh, Anton V. Persikov, and Marcus B. Noyes
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Interface (Java) ,Computational biology ,Biology ,Polymerase Chain Reaction ,Deep sequencing ,Genome engineering ,Domain (software engineering) ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Genomic library ,Gene Library ,030304 developmental biology ,Zinc finger ,0303 health sciences ,Binding Sites ,Computational Biology ,High-Throughput Nucleotide Sequencing ,Zinc Fingers ,Binding (Molecular Function) ,DNA-Binding Proteins ,Mutagenesis ,Proof of concept ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
The Cys2His2 zinc finger (ZF) is the most frequently found sequence-specific DNA-binding domain in eukaryotic proteins. The ZF’s modular protein–DNA interface has also served as a platform for genome engineering applications. Despite decades of intense study, a predictive understanding of the DNA-binding specificities of either natural or engineered ZF domains remains elusive. To help fill this gap, we developed an integrated experimental-computational approach to enrich and recover distinct groups of ZFs that bind common targets. To showcase the power of our approach, we built several large ZF libraries and demonstrated their excellent diversity. As proof of principle, we used one of these ZF libraries to select and recover thousands of ZFs that bind several 3-nt targets of interest. We were then able to computationally cluster these recovered ZFs to reveal several distinct classes of proteins, all recovered from a single selection, to bind the same target. Finally, for each target studied, we confirmed that one or more representative ZFs yield the desired specificity. In sum, the described approach enables comprehensive large-scale selection and characterization of ZF specificities and should be a great aid in furthering our understanding of the ZF domain. more...
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- 2013
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16. Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch
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Dana C. Nadler, David F. Savage, Jennifer A. Doudna, Avi I. Flamholz, Christof Fellmann, Brett T. Staahl, and Benjamin L. Oakes
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0301 basic medicine ,Protein design ,Protein domain ,PDZ domain ,Biomedical Engineering ,Bioengineering ,Computational biology ,Biology ,Protein Engineering ,Applied Microbiology and Biotechnology ,DNA-binding protein ,Genome ,Article ,Insertional mutagenesis ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Allosteric Regulation ,Protein Domains ,Insertional ,CRISPR-Associated Protein 9 ,MD Multidisciplinary ,CRISPR ,Site-Directed ,Clustered Regularly Interspaced Short Palindromic Repeats ,Genetics ,Binding Sites ,Cas9 ,Switch ,Endonucleases ,Mutagenesis, Insertional ,030104 developmental biology ,Genes ,Mutagenesis ,Mutagenesis, Site-Directed ,Molecular Medicine ,Genes, Switch ,030217 neurology & neurosurgery ,Biotechnology ,Protein Binding - Abstract
The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9. more...
- Published
- 2016
17. Copper modulates the phenotypic response of activated BV2 microglia through the release of nitric oxide
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Andrew J. Gow, Benjamin L. Oakes, Alba Rossi-George, and Chang-Jiang Guo
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Cancer Research ,Cell Survival ,Physiology ,Clinical Biochemistry ,Cell ,Fluorescent Antibody Technique ,Inflammation ,Biology ,Nitric Oxide ,Biochemistry ,Article ,Cell Line ,Nitric oxide ,Mice ,chemistry.chemical_compound ,medicine ,Animals ,Cytotoxic T cell ,Viability assay ,Microglia ,Neurodegeneration ,Neurodegenerative Diseases ,medicine.disease ,Cell biology ,Phenotype ,medicine.anatomical_structure ,chemistry ,Cell culture ,Inflammation Mediators ,medicine.symptom ,Copper - Abstract
Microglia are resident immune cells of the central nervous system. Their persistent activation in neurodegenerative diseases, traditionally attributed to neuronal dysfunction, may be due to a microglial failure to modulate the release of cytotoxic mediators such as nitric oxide (NO). The persistent activation of microglia with the subsequent release of NO vis-á-vis the accumulation of redox transition metals such as copper (Cu) in neurodegenerative diseases, prompted the hypothesis that copper would alter NO signaling by changing the redox environment of the cell and that, by altering the fate of NO, microglia would adopt a different phenotype. We have used the microglial cell model, BV2, to examine the effects of Cu(I) on NO production and activation as they have been shown to be phenotypically plastic. Our results show that cell viability is not affected by Cu(I) in BV2 microglia and that it has no effect on iNOS mRNA, protein expression and nitrite release. However, when LPS is added to Cu(I)-treated medium, nitrite release is abrogated while iNOS expression is not significantly altered. This effect is Cu(I)-specific and it is not observed with other non-redox metals, suggesting that Cu(I) modulates NO reactivity. Immunofluorescence analysis shows that the M1 (inflammatory) phenotype of BV2 microglia observed in response to LPS, is shifted to an M2 (adaptive) phenotype when Cu(I) is administered in combination with LPS. This same shift is not observed when iNOS function is inhibited by 1400W. In the present study we show that Cu(I) modulates the release of NO to the media, without altering iNOS expression, and produces a phenotypic changes in BV2 microglia. more...
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- 2012
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18. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing
- Author
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Marcus B. Noyes, Edward J. Rebar, Benjamin L. Oakes, Elizabeth F. Rowland, Jennifer S. Borchardt, Patrick Li, Danny F Xia, Lei Zhang, Jeffrey C. Miller, Irina Ankoudinova, and Denise J. Xu
- Subjects
0301 basic medicine ,Receptors, CCR5 ,Receptors, CCR2 ,Science ,General Physics and Astronomy ,Biology ,DNA-binding protein ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Homology (biology) ,Article ,Gene Expression Regulation, Enzymologic ,Genome engineering ,03 medical and health sciences ,Genome editing ,Genes, Reporter ,Peptide Library ,Animals ,Humans ,Gene ,Genetics ,Zinc finger ,Multidisciplinary ,Deoxyribonucleases ,Zinc Fingers ,General Chemistry ,Zinc finger nuclease ,body regions ,DNA-Binding Proteins ,030104 developmental biology - Abstract
Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity., Zinc finger nucleases have an established role in genome editing. Here, the authors report a strategy for identifying zinc finger nucleases that discriminate between desired targets and provide genome-wide specificity. more...
- Published
- 2016
19. Programmable RNA recognition and cleavage by CRISPR/Cas9
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Matias Kaplan, Samuel H. Sternberg, Mitchell R. O’Connell, Alexandra East-Seletsky, Benjamin L. Oakes, and Jennifer A. Doudna
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Cell Extracts ,Base pair ,General Science & Technology ,Messenger ,CRISPR-Associated Proteins ,Oligonucleotides ,Computational biology ,Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,MD Multidisciplinary ,Genetics ,CRISPR ,Humans ,RNA, Guide ,Clustered Regularly Interspaced Short Palindromic Repeats ,RNA, Messenger ,Kinetoplastida ,Nucleotide Motifs ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Base Sequence ,Cas9 ,Oligonucleotide ,RNA ,DNA ,Glyceraldehyde-3-Phosphate Dehydrogenase ,DNA binding site ,Protospacer adjacent motif ,chemistry ,Hela Cells ,Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ,CRISPR-Cas Systems ,Genetic Engineering ,030217 neurology & neurosurgery ,Guide ,HeLa Cells ,RNA, Guide, Kinetoplastida - Abstract
The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags. more...
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- 2014
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20. Protein engineering of Cas9 for enhanced function
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Benjamin L. Oakes, Dana C. Nadler, and David F. Savage
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Models, Molecular ,aTC ,Protein Conformation ,CRISPR-Associated Proteins ,PI ,PDZ Domains ,Protein Engineering ,Synthetic biology ,Genome editing ,Models ,HR ,ssDNA ,RFP ,CRISPR ,crRNA ,Cas9 ,Genetics ,SpCas9 ,LB ,sgRNA ,Biotechnology ,Biochemistry & Molecular Biology ,Streptococcus pyogenes ,1.1 Normal biological development and functioning ,PDZ domain ,Molecular Sequence Data ,FACS ,Bioengineering ,Computational biology ,BH ,Biology ,GFP ,Article ,dCas9 ,Nuclease ,tracrRNA ,Underpinning research ,Gene activation/repression ,IPTG ,NUC ,Deoxyribonuclease I ,PDZ ,SOB ,Amino Acid Sequence ,SOC ,NHEJ ,Trans-activating crRNA ,Bacteria ,Base Sequence ,REC ,Molecular ,Protein engineering ,DNA ,Fusion protein ,WT dCas9 ,IT dCas9 ,EM ,Mutation ,PAM ,RNA ,Generic health relevance ,Biochemistry and Cell Biology ,CRISPR-Cas Systems - Abstract
CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complimentary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted directly into the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome modifying tools. more...
- Published
- 2014
21. Rapid synthesis and screening of chemically activated transcription factors with GFP-based reporters
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R. Scott McIsaac, David Botstein, Benjamin L. Oakes, and Marcus B. Noyes
- Subjects
Zif268 ,General Chemical Engineering ,Green Fluorescent Proteins ,Molecular Sequence Data ,Artificial transcription factor ,Biology ,Protein Engineering ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Synthetic biology ,0302 clinical medicine ,Transcription (biology) ,Yeasts ,transcription factors ,Gene expression ,Genetics ,Humans ,URA3 ,artificial transcription factors ,Transcription factor ,030304 developmental biology ,0303 health sciences ,General Immunology and Microbiology ,Base Sequence ,General Neuroscience ,Flow Cytometry ,zinc fingers ,Peptide Fragments ,Cell biology ,Connexin 43 ,Synthetic Biology ,Issue 81 ,Homologous recombination ,transcription ,030217 neurology & neurosurgery ,Binding domain ,Plasmids - Abstract
Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided. more...
- Published
- 2013
22. Binding Site Selection and Cleavage Activity of Zinc Finger Nucleases Targeting Gaucher Mutation
- Author
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Elizabeth A. Rowland, Marcus B. Noyes, Benjamin L. Oakes, and Sanjeev Dahal
- Subjects
Chemistry ,Mutation (genetic algorithm) ,Genetics ,Binding site ,Cleavage (embryo) ,Molecular Biology ,Biochemistry ,Zinc finger nuclease ,Molecular biology ,Selection (genetic algorithm) ,Biotechnology - Published
- 2013
- Full Text
- View/download PDF
23. 564. Finding the Needle in a Haystack: Precise Genome Editing With a Multi-Reporter Selection Systems
- Author
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Benjamin L. Oakes, Marcus B. Noyes, Jeffrey C. Miller, Danny F Xia, Edward J. Rebar, and Elizabeth F. Rowland
- Subjects
Pharmacology ,Genetics ,Zinc finger ,Nuclease ,biology ,ved/biology ,ved/biology.organism_classification_rank.species ,Genome ,Zinc finger nuclease ,Genome editing ,Drug Discovery ,biology.protein ,Molecular Medicine ,Indel ,Model organism ,Molecular Biology ,Gene - Abstract
Designer nucleases have revolutionized the study of common model organisms by enabling the simple and efficient manipulation of specified genomic sequences. However, therapeutic application of this technology may be limited by predictable and unanticipated off-target lesions that may be prevalent with all nuclease platforms to lesser and greater extents. This limitation may become even more problematic when desired targets are homologous to other genome sequences. Here we report the development of a multi-reporter selection system for the generation of nucleases that can discriminate between highly homologous targets. We have applied this system to create zinc finger nuclease (ZFNs) that can discriminate between highly homologous sequences from the CCR5 and CCR2 genes. These nucleases were tested in human cell culture and demonstrate strong on-target activity (≈10-60% indel frequency) with minimal or no nuclease activity at CCR2. Further, an unbiased screen of off-target activity demonstrates excellent genome-wide fidelity. In one case, the nuclease pair results in only one off-target with greater than 0.1% indel frequency (0.37%) and an aggregate off-target activity of 0.5%. This system offers a simple approach to generate nucleases, zinc finger or otherwise, with exceptional genome-wide fidelity regardless of target. more...
- Published
- 2015
- Full Text
- View/download PDF
24. Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast
- Author
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David Botstein, Benjamin L. Oakes, Xin Wang, Krysta A. Dummit, Marcus B. Noyes, and R. Scott McIsaac
- Subjects
Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Recombinant Fusion Proteins ,Gene regulatory network ,Saccharomyces cerevisiae ,Biology ,Gene product ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Gene expression ,Genetics ,Animals ,Humans ,Gene Regulatory Networks ,Transcription factor ,Gene ,030304 developmental biology ,Cell Proliferation ,Early Growth Response Protein 1 ,Regulation of gene expression ,Zinc finger ,0303 health sciences ,Herpes simplex virus protein vmw65 ,Binding Sites ,Estradiol ,Herpes Simplex Virus Protein Vmw65 ,Zinc Fingers ,Cell biology ,Protein Structure, Tertiary ,Basic-Leucine Zipper Transcription Factors ,Gene Expression Regulation ,Receptors, Estrogen ,Methods Online ,Genome, Fungal ,Genetic Engineering ,030217 neurology & neurosurgery - Abstract
A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys(2)His(2) zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p. more...
- Published
- 2012
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