11 results on '"Addison V. Wright"'
Search Results
2. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling
- Author
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Addison V. Wright, Joshua C Cofsky, Zev Bryant, Ivan Ivanov, Jennifer A. Doudna, and Kevin D Palacio Aris
- Subjects
1.1 Normal biological development and functioning ,CRISPR-Associated Proteins ,Bioengineering ,Genome ,chemistry.chemical_compound ,Underpinning research ,Cleave ,Genetics ,Guide RNA ,DNA Cleavage ,Kinetoplastida ,Base Pairing ,Gene Editing ,Nuclease ,Multidisciplinary ,biology ,Chemistry ,Cas9 ,Energy landscape ,DNA ,Biological Sciences ,Endonucleases ,torque spectroscopy ,Biophysics ,biology.protein ,DNA supercoil ,RNA ,Generic health relevance ,CRISPR-Cas Systems ,R-Loop Structures ,magnetic tweezers ,Guide ,RNA, Guide, Kinetoplastida ,Biotechnology - Abstract
The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.
- Published
- 2020
3. Structures of the CRISPR genome integration complex
- Author
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Gavin J. Knott, Jun-Jie Liu, Eva Nogales, Kevin W. Doxzen, Jennifer A. Doudna, and Addison V. Wright
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DNA, Bacterial ,Integration Host Factors ,0301 basic medicine ,General Science & Technology ,1.1 Normal biological development and functioning ,CRISPR-Associated Proteins ,Bacteremia ,Locus (genetics) ,Crystallography, X-Ray ,Genome ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Underpinning research ,Catalytic Domain ,Medicine and Health Sciences ,Genetics ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Trans-activating crRNA ,CRISPR interference ,Endodeoxyribonucleases ,Crystallography ,Multidisciplinary ,Integrases ,biology ,Cas9 ,Escherichia coli Proteins ,Cryoelectron Microscopy ,Bacterial ,DNA ,Endonucleases ,Integrase ,030104 developmental biology ,chemistry ,Mutation ,X-Ray ,biology.protein ,Generic health relevance ,CRISPR-Cas Systems - Abstract
© 2017, American Association for the Advancement of Science. All rights reserved. CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo–electron microscopy structure of the full CRISPR locus integration complex, including the accessory protein IHF (integration host factor). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity.
- Published
- 2017
4. Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response
- Author
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Marija Vucelja, Addison V. Wright, Luciano A. Marraffini, Robert Heler, David Bikard, and Jennifer A. Doudna
- Subjects
0301 basic medicine ,Staphylococcus aureus ,Time Factors ,Genotype ,CRISPR-Associated Proteins ,030106 microbiology ,adaptation ,Adaptive Immunity ,Biology ,Medical and Health Sciences ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Plasmid ,bacteriophage ,Bacterial Proteins ,CRISPR-Associated Protein 9 ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Viral ,CRISPR-Cas ,Cas9 ,Molecular Biology ,Genetics ,Trans-activating crRNA ,CRISPR interference ,Intergenic ,High-Throughput Nucleotide Sequencing ,DNA ,Cell Biology ,Biological Sciences ,Endonucleases ,Acquired immune system ,Phenotype ,030104 developmental biology ,chemistry ,DNA, Viral ,Host-Pathogen Interactions ,Mutation ,CRISPR Loci ,horizontal gene transfer ,DNA, Intergenic ,spacer acquisition ,CRISPR-Cas Systems ,Developmental Biology - Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the inavding DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, which increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application.
- Published
- 2017
5. Protecting genome integrity during CRISPR immune adaptation
- Author
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Addison V. Wright and Jennifer A. Doudna
- Subjects
0301 basic medicine ,Streptococcus pyogenes ,CRISPR-Associated Proteins ,Genome ,DNA sequencing ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA Integration ,Molecular Biology ,Genetics ,Base Sequence ,Integrases ,biology ,Cas9 ,DNA ,Acquired immune system ,Integrase ,030104 developmental biology ,chemistry ,biology.protein ,CRISPR-Cas Systems ,Genome, Bacterial - Abstract
Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1-Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.
- Published
- 2016
6. Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering
- Author
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Addison V. Wright, Jennifer A. Doudna, and James K. Nuñez
- Subjects
0301 basic medicine ,Genetics ,Bacteria ,Biochemistry, Genetics and Molecular Biology(all) ,CRISPR-Associated Proteins ,Computational biology ,Plants ,Biology ,Endonucleases ,Archaea ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Genome engineering ,03 medical and health sciences ,030104 developmental biology ,Viral genetics ,Plasmid ,DNA, Viral ,Nucleic acid ,Animals ,Humans ,CRISPR ,CRISPR-Cas Systems ,Genetic Engineering ,Function (biology) - Abstract
Bacteria and archaea possess a range of defense mechanisms to combat plasmids and viral infections. Unique among these are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems, which provide adaptive immunity against foreign nucleic acids. CRISPR systems function by acquiring genetic records of invaders to facilitate robust interference upon reinfection. In this Review, we discuss recent advances in understanding the diverse mechanisms by which Cas proteins respond to foreign nucleic acids and how these systems have been harnessed for precision genome manipulation in a wide array of organisms.
- Published
- 2016
7. Spacer Acquisition Rates Determine the Immunological Diversity of the Type II CRISPR-Cas Immune Response
- Author
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Marija Vucelja, Addison V. Wright, Robert Heler, Jennifer A. Doudna, and Luciano A. Marraffini
- Subjects
Small RNA ,Staphylococcus aureus ,Streptococcus pyogenes ,Population ,medicine.disease_cause ,Microbiology ,Genome ,Article ,Bacteriophage ,Evolution, Molecular ,03 medical and health sciences ,0302 clinical medicine ,Virology ,medicine ,CRISPR ,education ,030304 developmental biology ,Genetics ,0303 health sciences ,education.field_of_study ,biology ,Cas9 ,biology.organism_classification ,Acquired immune system ,DNA, Viral ,Parasitology ,DNA, Intergenic ,CRISPR-Cas Systems ,Staphylococcus Phages ,030217 neurology & neurosurgery - Abstract
CRISPR-Cas systems provide acquired immunity in prokaryotes. Upon infection, short sequences from the phage genome, known as spacers, are inserted between the CRISPR repeats. Spacers are transcribed into small RNA molecules that guide nucleases to their targets. The forces that shape the distribution of newly acquired spacers, which is observed to be uneven, are poorly understood. We studied the spacer patterns that arise after phage infection of Staphylococcus aureus harboring the Streptococcus pyogenes type II-A CRISPR-Cas system. We observed that spacer patterns are established early during the CRISPR-Cas immune response and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The rate of spacer acquisition depended on sequence elements within the spacer, which in turn determined the abundance of different spacers within the adapted population. Our results reveal how the two main forces of the CRISPR-Cas immune response, acquisition and targeting, affect the generation of immunological diversity.
- Published
- 2018
8. Keeping CRISPR under control: how bacteria fight viruses without harming themselves
- Author
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Addison V. Wright
- Subjects
biology ,CRISPR ,biology.organism_classification ,Bacteria ,Microbiology - Published
- 2018
9. Rational design of a split-Cas9 enzyme complex
- Author
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Jennifer A. Doudna, Jack E. Kornfeld, Jorge A Bardales, Brett T. Staahl, David W. Taylor, Addison V. Wright, and Samuel H. Sternberg
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Enzyme complex ,Transcription, Genetic ,Streptococcus pyogenes ,Electrophoretic Mobility Shift Assay ,chemistry.chemical_compound ,Genetic ,MD Multidisciplinary ,Transcriptional regulation ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Guide RNA ,Genetics ,Nuclease ,Multidisciplinary ,biology ,split enzyme ,Cas9 ,Hydrolysis ,RNA ,DNA ,Biological Sciences ,Cell biology ,chemistry ,biology.protein ,Nucleic Acid Conformation ,CRISPR-Cas9 ,genome engineering ,Transcription - Abstract
Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.
- Published
- 2015
10. Cas1–Cas2 complex formation mediates spacer acquisition during CRISPR–Cas adaptive immunity
- Author
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Jonas Noeske, James K. Nuñez, Christopher W. Davies, Philip J. Kranzusch, Addison V. Wright, and Jennifer A. Doudna
- Subjects
Models, Molecular ,Mutant ,CRISPR-Associated Proteins ,Locus (genetics) ,Computational biology ,Biology ,Adaptive Immunity ,medicine.disease_cause ,Crystallography, X-Ray ,Article ,chemistry.chemical_compound ,Protein structure ,Structural Biology ,Immunity ,Medicine and Health Sciences ,medicine ,Escherichia coli ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Molecular Biology ,Genetics ,Endodeoxyribonucleases ,Escherichia coli Proteins ,Acquired immune system ,Endonucleases ,Protein Structure, Tertiary ,chemistry ,CRISPR-Cas Systems ,DNA - Abstract
The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.
- Published
- 2014
11. A Functional Mini-Integrase in a Two-Protein Type V-C CRISPR System
- Author
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Joy Y. Wang, David Burstein, Lucas B. Harrington, Jillian F. Banfield, Nikos C. Kyrpides, Jennifer A. Doudna, David Paez-Espino, Anthony T. Iavarone, and Addison V. Wright
- Subjects
DNA, Bacterial ,Base pair ,CRISPR-Associated Proteins ,Locus (genetics) ,Computational biology ,Biology ,Article ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Escherichia coli ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,DNA Integration ,Nucleotide Motifs ,Base Pairing ,Molecular Biology ,Gene ,030304 developmental biology ,Gene Editing ,0303 health sciences ,Endodeoxyribonucleases ,Integrases ,Escherichia coli Proteins ,Gene Expression Regulation, Bacterial ,Cell Biology ,Endonucleases ,Integrase ,A-site ,chemistry ,biology.protein ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,DNA - Abstract
CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.
- Published
- 2019
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