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Sheena E. Radford - One of the best experts on this subject based on the ideXlab platform.
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Dissecting key residues in folding and stability of the Bacterial Immunity protein 7
Protein engineering design & selection : PEDS, 2011Co-Authors: Stuart Knowling, Alice I. Bartlett, Sheena E. RadfordAbstract:The small four-helix Immunity protein, Im7, has previously been shown to fold via a compact intermediate containing three of the four native helices. The short, six-residue helix III only docks onto the developing Im7 structure after the rate-limiting second transition state has been traversed. Previous work demonstrated that mutation of the helix III sequence can be used to trap the protein in the on-pathway intermediate ensemble at equilibrium. Here the role played by individual residues in the native helix III sequence in locking Im7 into a stable native structure is further examined. This work commenced with an Im7 sequence trapped in the partially folded state by substitution of the six residues in helix III with a polyglycine sequence. Biophysical analysis of variants in which individual residues from the native helix III sequence, and combinations of these residues, were introduced into this background demonstrated a critical requirement for three residues, Leu 53, Ile 54 and Tyr 55, to lock Im7 into its unique native structure. The results demonstrate a stringent constraint on the evolution of the Im7 helix III sequence rationalizing its high-sequence identity in the fold family. Thus, Leu 53 and Ile 54 provide crucial stabilizing interactions in the hydrophobic core of native Im7, while Tyr 55 is required for both stability and function. In contrast, Tyr 56 is critical for colicin binding and has no role in maintaining a stable native fold.
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Perturbing the folding energy landscape of the Bacterial Immunity protein Im7 by site-specific N-linked glycosylation
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Mark M. Chen, Alice I. Bartlett, Paul S. Nerenberg, Claire T. Friel, Christian P. R. Hackenberger, Collin M. Stultz, Sheena E. Radford, Barbara ImperialiAbstract:N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the Bacterial Immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The ΔΔG° values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol(-1). In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.
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the mechanism of folding of im7 reveals competition between functional and kinetic evolutionary constraints
Nature Structural & Molecular Biology, 2009Co-Authors: Claire T. Friel, Joerg Gsponer, Michele Vendruscolo, Alastair D Smith, Sheena E. RadfordAbstract:Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the Bacterial Immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.
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The effect of increasing the stability of non-native interactions on the folding landscape of the Bacterial Immunity protein Im9.
Journal of molecular biology, 2007Co-Authors: Victoria L. Morton, Claire T. Friel, Emanuele Paci, Lucy R. Allen, Sheena E. RadfordAbstract:How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the Bacterial Immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Φ-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in Immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Φ-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the Immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.
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NMR Analysis of the Conformational Properties of the Trapped on-pathway Folding Intermediate of the Bacterial Immunity Protein Im7
Journal of molecular biology, 2006Co-Authors: Sara B.-m. Whittaker, Sheena E. Radford, Graham R. Spence, J. Günter Grossmann, Geoffrey R. MooreAbstract:Previous work shows that the transiently populated, on-pathway intermediate in Im7 folding contains three of the four native α-helices docked around a core stabilised by native and non-native interactions. To determine the structure and dynamic properties of this species in more detail, we have used protein engineering to trap the intermediate at equilibrium and analysed the resulting proteins using NMR spectroscopy and small angle X-ray scattering. Four variants were created. In L53AI54A, two hydrophobic residues within helix III are truncated, preventing helix III from docking stably onto the developing hydrophobic core. In two other variants, the six residues encompassing the native helix III were replaced with three (H3G3) or six (H3G6) glycine residues. In the fourth variant, YY, two native tyrosine residues (Tyr55 and Tyr56) were re-introduced into H3G6 to examine their role in determining the properties of the intermediate ensemble. All four variants show variable peak intensities and broad peak widths, consistent with these proteins being conformationally dynamic. Chemical shift analyses demonstrated that L53AI54A and YY contain native-like secondary structure in helices I and IV, while helix II is partly formed and helix III is absent. Lack of NOEs and rapid NH exchange for L53AI54A, combined with detailed analysis of the backbone dynamics, indicated that the hydrophobic core of this variant is not uniquely structured, but fluctuates on the NMR timescale. The results demonstrate that though much of the native-like secondary structure of Im7 is present in the variants, their hydrophobic cores remain relatively fluid. The comparison of H3G3/H3G6 and L53AI54A/YY suggests that Tyr55 and/or Tyr56 interact with the three-helix core, leading other residues in this region of the protein to dock with the core as folding progresses. In this respect, the three-helix bundle acts as a template for formation of helix III and the creation of the native fold.
Jennifer A. Doudna - One of the best experts on this subject based on the ideXlab platform.
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rna binding and hepn nuclease activation are decoupled in crispr cas13a
bioRxiv, 2017Co-Authors: Akshay Tambe, Jennifer A. Doudna, Alexandra Eastseletsky, Gavin J Knott, Mitchell R OconnellAbstract:CRISPR-Cas13a is a CRISPR RNA (crRNA)-guided, RNA-activated ribonuclease (RNase) involved in Bacterial adaptive Immunity and shows promise as a useful tool for RNA detection, RNA imaging and RNA regulation. However, the relationship between Cas13a9s crRNA-mediated RNA-activator binding and HEPN nuclease activation is not well understood. Using a comprehensive sequencing-based strategy coupled with in vitro biochemistry, we find that Cas13a9s crRNA-guided RNA binding affinity and HEPN nuclease activity are differentially affected by the number and position of mismatches between the crRNA and activator-RNA. Unexpectedly, activator-RNAs with the tightest binding affinity are not perfectly complementary to the crRNA, but rather contain mismatches at specific positions. However, despite tight binding to Cas13a, these RNA-activators do not activate Cas13a9s HEPN nuclease. Conversely, a subset of mismatched, weakly-bound activator-RNAs are able to maximally activate Cas13a9s HEPN nuclease. These results demonstrate that RNA-activator binding and target cleavage by Cas13a are decoupled, highlighting a complex specificity landscape that allows for plasticity in the precise RNA sequence required by Cas13a for RNA binding and HEPN nuclease activation. Taken together, our findings underscore a need to consider the range of effects off-target recognition has on Cas13a9s RNA binding and cleavage behavior in both Bacterial Immunity and RNA-targeting tool development.
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a Bacterial argonaute with noncanonical guide rna specificity
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Jennifer A. Doudna, Emine Kaya, Kevin W Doxzen, Kilian R Knoll, Ross C Wilson, Steven C Strutt, Philip J KranzuschAbstract:Eukaryotic Argonaute proteins induce gene silencing by small RNA-guided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] Bacterial Immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-A resolution crystal structure of an MpAgo–RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPR-cas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide.
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rna guided genetic silencing systems in bacteria and archaea
Nature, 2012Co-Authors: Blake Wiedenheft, Samuel H Sternberg, Jennifer A. DoudnaAbstract:Clustered regularly interspaced short palindromic repeat (CRISPR) are essential components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. Similar to RNA interference (RNAi) pathways in eukaryotes, CRISPR-mediated immune systems rely on small RNAs for sequence-specific detection and silencing of foreign nucleic acids, including viruses and plasmids. However, the mechanism of RNA-based Bacterial Immunity is distinct from RNAi. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems.
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A programmable dual-RNA-guided DNA endonuclease in adaptive Bacterial Immunity
Science, 2012Co-Authors: Martin Jinek, Ines Fonfara, Krzysztof Chylinski, Michelle Hauer, Jennifer A. Doudna, Emmanuelle CharpentierAbstract:Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive Immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
Claire T. Friel - One of the best experts on this subject based on the ideXlab platform.
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Perturbing the folding energy landscape of the Bacterial Immunity protein Im7 by site-specific N-linked glycosylation
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Mark M. Chen, Alice I. Bartlett, Paul S. Nerenberg, Claire T. Friel, Christian P. R. Hackenberger, Collin M. Stultz, Sheena E. Radford, Barbara ImperialiAbstract:N-linked glycosylation modulates protein folding and stability through a variety of mechanisms. As such there is considerable interest in the development of general rules to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in the design and development of modified proteins with advantageous properties. In this study, expressed protein ligation is used to create site-specifically glycosylated variants of the Bacterial Immunity protein Im7 modified with the chitobiose disaccharide (GlcNAc-GlcNAc). Glycans were introduced at seven solvent exposed sites within the Im7 sequence and the kinetic and thermodynamic consequences of N-linked glycosylation analyzed. The ΔΔG° values for glycan incorporation were found to range from +5.2 to -3.8 kJ·mol(-1). In several cases, glycosylation influences folding by modulating the local conformational preferences of the glycosylated sequence. These locally mediated effects are most prominent in the center of α-helices where glycosylation negatively effects folding and in compact turn motifs between segments of ordered secondary structure where glycosylation promotes folding and enhances the overall stability of the native protein. The studies also provide insight into why glycosylation is commonly identified at the transition between different types of secondary structure and when glycosylation may be used to elaborate protein structure to protect disordered sequences from proteolysis or immune system recognition.
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the mechanism of folding of im7 reveals competition between functional and kinetic evolutionary constraints
Nature Structural & Molecular Biology, 2009Co-Authors: Claire T. Friel, Joerg Gsponer, Michele Vendruscolo, Alastair D Smith, Sheena E. RadfordAbstract:Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the Bacterial Immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.
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The effect of increasing the stability of non-native interactions on the folding landscape of the Bacterial Immunity protein Im9.
Journal of molecular biology, 2007Co-Authors: Victoria L. Morton, Claire T. Friel, Emanuele Paci, Lucy R. Allen, Sheena E. RadfordAbstract:How stabilising non-native interactions influence protein folding energy landscapes is currently not well understood: such interactions could speed folding by reducing the conformational search to the native state, or could slow folding by increasing ruggedness. Here, we examine the influence of non-native interactions in the folding process of the Bacterial Immunity protein Im9, by exploiting our ability to manipulate the stability of the intermediate and rate-limiting transition state (TS) in the folding of this protein by minor alteration of its sequence or changes in solvent conditions. By analysing the properties of these species using Φ-value analysis, and exploration of the structural properties of the TS ensemble using molecular dynamics simulations, we demonstrate the importance of non-native interactions in Immunity protein folding and demonstrate that the rate-limiting step involves partial reorganisation of these interactions as the TS ensemble is traversed. Moreover, we show that increasing the contribution to stability made by non-native interactions results in an increase in Φ-values of the TS ensemble without altering its structural properties or solvent-accessible surface area. The data suggest that the Immunity proteins fold on multiple, but closely related, micropathways, resulting in a heterogeneous TS ensemble that responds subtly to mutation or changes in the solvent conditions. Thus, altering the relative strength of native and non-native interactions influences the search to the native state by restricting the pathways through the folding energy landscape.
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Helix stability and hydrophobicity in the folding mechanism of the Bacterial Immunity protein Im9
Protein engineering design & selection : PEDS, 2005Co-Authors: Susanne Cranz-mileva, Claire T. Friel, Sheena E. RadfordAbstract:Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.
Andreas Kupz - One of the best experts on this subject based on the ideXlab platform.
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in vivo ifn γ secretion by nk cells in response to salmonella typhimurium requires nlrc4 inflammasomes
PLOS ONE, 2014Co-Authors: Andreas Kupz, Roy Curtiss, Sammy Bedoui, Richard A StrugnellAbstract:Natural killer (NK) cells are a critical part of the innate immune defense against viral infections and for the control of tumors. Much less is known about how NK cells contribute to anti-Bacterial Immunity. NK cell-produced interferon gamma (IFN-γ) contributes to the control of early exponential replication of Bacterial pathogens, however the regulation of these events remains poorly resolved. Using a mouse model of invasive Salmonellosis, here we report that the activation of the intracellular danger sensor NLRC4 by Salmonella-derived flagellin within CD11c+ cells regulates early IFN-γ secretion by NK cells through the provision of interleukin 18 (IL-18), independently of Toll-like receptor (TLR)-signaling. Although IL18-signalling deficient NK cells improved host protection during S. Typhimurium infection, this increased resistance was inferior to that provided by wild-type NK cells. These findings suggest that although NLRC4 inflammasome-driven secretion of IL18 serves as a potent activator of NK cell mediated IFN-γ secretion, IL18-independent NK cell-mediated mechanisms of IFN-γ secretion contribute to in vivo control of Salmonella replication.
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nlrc4 inflammasomes in dendritic cells regulate noncognate effector function by memory cd8 t cells
Nature Immunology, 2012Co-Authors: Andreas Kupz, Greta Guarda, Thomas Gebhardt, Leif E Sander, Kirsty R Short, Dimitri A Diavatopoulos, Odilia L C Wijburg, Hanwei Cao, Jason WaithmanAbstract:Memory T cells exert antigen-independent effector functions, but how these responses are regulated is unclear. We discovered an in vivo link between flagellin-induced NLRC4 inflammasome activation in splenic dendritic cells (DCs) and host protective interferon-γ (IFN-γ) secretion by noncognate memory CD8(+) T cells, which could be activated by Salmonella enterica serovar Typhimurium, Yersinia pseudotuberculosis and Pseudomonas aeruginosa. We show that CD8α(+) DCs were particularly efficient at sensing Bacterial flagellin through NLRC4 inflammasomes. Although this activation released interleukin 18 (IL-18) and IL-1β, only IL-18 was required for IFN-γ production by memory CD8(+) T cells. Conversely, only the release of IL-1β, but not IL-18, depended on priming signals mediated by Toll-like receptors. These findings provide a comprehensive mechanistic framework for the regulation of noncognate memory T cell responses during Bacterial Immunity.
Luciano A Marraffini - One of the best experts on this subject based on the ideXlab platform.
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enhanced Bacterial Immunity and mammalian genome editing via rna polymerase mediated dislodging of cas9 from double strand dna breaks
Molecular Cell, 2018Co-Authors: Ryan Clarke, Robert Heler, Matthew S Macdougall, Alejandro Chavez, Maureen Regan, Leslyn A Hanakahi, George M Church, Luciano A Marraffini, Bradley J MerrillAbstract:Summary The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing Bacterial Immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.
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enhanced Bacterial Immunity and mammalian genome editing via rna polymerase mediated dislodging of cas9 from double strand dna breaks
bioRxiv, 2018Co-Authors: Ryan Clarke, Robert Heler, Matthew S Macdougall, Alejandro Chavez, Maureen Regan, Leslyn A Hanakahi, George M Church, Luciano A Marraffini, Bradley J MerrillAbstract:The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing Bacterial Immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.
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CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems
Annual review of microbiology, 2015Co-Authors: Wenyan Jiang, Luciano A MarraffiniAbstract:Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most n...