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CRISPR

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Jennifer A Doudna - One of the best experts on this subject based on the ideXlab platform.

  • two distinct rnase activities of CRISPR c2c2 enable guide rna processing and rna detection
    Nature, 2016
    Co-Authors: Alexandra Eastseletsky, Mitchell R Oconnell, Spencer C Knight, David E Burstein, Robert Tjian, Jamie H D Cate, Jennifer A Doudna
    Abstract:

    Bacterial adaptive immune systems use CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. Although most prokaryotic adaptive immune systems generally target DNA substrates, type III and VI CRISPR systems direct interference complexes against single-stranded RNA substrates. In type VI systems, the single-subunit C2c2 protein functions as an RNA-guided RNA endonuclease (RNase). How this enzyme acquires mature CRISPR RNAs (crRNAs) that are essential for immune surveillance and how it carries out crRNA-mediated RNA cleavage remain unclear. Here we show that bacterial C2c2 possesses a unique RNase activity responsible for CRISPR RNA maturation that is distinct from its RNA-activated single-stranded RNA degradation activity. These dual RNase functions are chemically and mechanistically different from each other and from the crRNA-processing behaviour of the evolutionarily unrelated CRISPR enzyme Cpf1 (ref. 11). The two RNase activities of C2c2 enable multiplexed processing and loading of guide RNAs that in turn allow sensitive detection of cellular transcripts.

  • CRISPR mediated modular rna guided regulation of transcription in eukaryotes
    Cell, 2013
    Co-Authors: Luke A. Gilbert, Leonardo Morsut, Zairan Liu, Sandra E. Torres, Onn Brandman, Evan H. Whitehead, Matthew H. Larson, Noam Sternginossar, Gloria A Brar, Jennifer A Doudna
    Abstract:

    Summary The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

  • repurposing CRISPR as an rna guided platform for sequence specific control of gene expression
    Cell, 2013
    Co-Authors: Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jonathan S. Weissman, Adam Paul Arkin, Jennifer A Doudna
    Abstract:

    SUMMARY Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

  • Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression
    Cell, 2013
    Co-Authors: Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jonathan S. Weissman, Adam Paul Arkin, Jennifer A Doudna, Wendell A. Lim
    Abstract:

    Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-γuided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-γuided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale. © 2013 Elsevier Inc.

  • XCRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes
    Cell, 2013
    Co-Authors: Luke A. Gilbert, Leonardo Morsut, Zairan Liu, Sandra E. Torres, Onn Brandman, Evan H. Whitehead, Noam Stern-ginossar, Matthew H. Larson, Gloria A Brar, Jennifer A Doudna
    Abstract:

    The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells. © 2013 Elsevier Inc.

Sylvain Moineau - One of the best experts on this subject based on the ideXlab platform.

  • An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9.
    Nature microbiology, 2017
    Co-Authors: Alexander P. Hynes, Dennis A Romero, Philippe Horvath, Christophe Fremaux, Geneviève M. Rousseau, Marie-laurence Lemay, Sylvain Moineau
    Abstract:

    The CRISPR–Cas system owes its utility as a genome-editing tool to its origin as a prokaryotic immune system. The first demonstration of its activity against bacterial viruses (phages) is also the first record of phages evading that immunity 1 . This evasion can be due to point mutations 1 , large-scale deletions 2 , DNA modifications 3 , or phage-encoded proteins that interfere with the CRISPR–Cas system, known as anti-CRISPRs (Acrs) 4 . The latter are of biotechnological interest, as Acrs can serve as off switches for CRISPR-based genome editing 5 . Every Acr characterized to date originated from temperate phages, genomic islands, or prophages 4–8 , and shared properties with the first Acr discovered. Here, with a phage-oriented approach, we have identified an unrelated Acr in a virulent phage of Streptococcus thermophilus. In challenging a S. thermophilus strain CRISPR-immunized against a set of virulent phages, we found one that evaded the CRISPR-encoded immunity >40,000× more often than the others. Through systematic cloning of its genes, we identified an Acr solely responsible for the abolished immunity. We extended our findings by demonstrating activity in another S. thermophilus strain, against unrelated phages, and in another bacterial genus immunized using the heterologous SpCas9 system favoured for genome editing. This Acr completely abolishes SpCas9-mediated immunity in our assays.

  • the CRISPR cas bacterial immune system cleaves bacteriophage and plasmid dna
    Nature, 2010
    Co-Authors: Josiane E. Garneau, Marie-Ève Dupuis, Manuela Villion, Dennis A Romero, Patrick Boyaval, Philippe Horvath, Alfonso H Magadán, Rodolphe Barrangou, Christophe Fremaux, Sylvain Moineau
    Abstract:

    Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains. CRISPR/Cas is a microbial immune system that is known to protect bacteria from viral infection. It is now shown that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA of both viral and plasmid origin. The system seems remarkably adapted to this end, and it is thought that CRISPR/Cas could be used to naturally generate safer and more robust bacteria that are resistant to the acquisition and spread of antibiotic resistance genes. CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.

  • CRISPR cas system and its role in phage bacteria interactions
    Annual Review of Microbiology, 2010
    Co-Authors: Hélène Deveau, Josiane E. Garneau, Sylvain Moineau
    Abstract:

    Clustered regularly interspaced short palindromic repeats (CRISPRs) along with Cas proteins is a widespread system across bacteria and archaea that causes interference against foreign nucleic acids. The CRISPR/Cas system acts in at least two general stages: the adaptation stage, where the cell acquires new spacer sequences derived from foreign DNA, and the interference stage, which uses the recently acquired spacers to target and cleave invasive nucleic acid. The CRISPR/Cas system participates in a constant evolutionary battle between phages and bacteria through addition or deletion of spacers in host cells and mutations or deletion in phage genomes. This review describes the recent progress made in this fast-expanding field.

  • The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
    Nature, 2010
    Co-Authors: Josiane E. Garneau, Marie-Ève Dupuis, Manuela Villion, Dennis A Romero, Patrick Boyaval, Philippe Horvath, Alfonso H Magadán, Rodolphe Barrangou, Christophe Fremaux, Sylvain Moineau
    Abstract:

    Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

  • diversity activity and evolution of CRISPR loci in streptococcus thermophilus
    Journal of Bacteriology, 2008
    Co-Authors: Philippe Horvath, Hélène Deveau, Dennis A Romero, Patrick Boyaval, Anneclaire Coutemonvoisin, Melissa Richards, Sylvain Moineau, Christophe Fremaux, Rodolphe Barrangou
    Abstract:

    Clustered regularly interspaced short palindromic repeats (CRISPR) are hypervariable loci widely distributed in prokaryotes that provide acquired immunity against foreign genetic elements. Here, we characterize a novel Streptococcus thermophilus locus, CRISPR3, and experimentally demonstrate its ability to integrate novel spacers in response to bacteriophage. Also, we analyze CRISPR diversity and activity across three distinct CRISPR loci in several S. thermophilus strains. We show that both CRISPR repeats and cas genes are locus specific and functionally coupled. A total of 124 strains were studied, and 109 unique spacer arrangements were observed across the three CRISPR loci. Overall, 3,626 spacers were analyzed, including 2,829 for CRISPR1 (782 unique), 173 for CRISPR2 (16 unique), and 624 for CRISPR3 (154 unique). Sequence analysis of the spacers revealed homology and identity to phage sequences (77%), plasmid sequences (16%), and S. thermophilus chromosomal sequences (7%). Polymorphisms were observed for the CRISPR repeats, CRISPR spacers, cas genes, CRISPR motif, locus architecture, and specific sequence content. Interestingly, CRISPR loci evolved both via polarized addition of novel spacers after exposure to foreign genetic elements and via internal deletion of spacers. We hypothesize that the level of diversity is correlated with relative CRISPR activity and propose that the activity is highest for CRISPR1, followed by CRISPR3, while CRISPR2 may be degenerate. Globally, the dynamic nature of CRISPR loci might prove valuable for typing and comparative analyses of strains and microbial populations. Also, CRISPRs provide critical insights into the relationships between prokaryotes and their environments, notably the coevolution of host and viral genomes.

Luke A. Gilbert - One of the best experts on this subject based on the ideXlab platform.

  • a multiplexed single cell CRISPR screening platform enables systematic dissection of the unfolded protein response
    Cell, 2016
    Co-Authors: Britt Adamson, Jacqueline E. Villalta, Max A. Horlbeck, Luke A. Gilbert, Yuwen Chen, Thomas M Norman, Marco Jost, James K Nunez, Marco Y Hein, Andrew N Gray
    Abstract:

    Summary Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

  • abstract a2 56 cancer specific synthetic lethality and network rewiring elucidated by genetic interaction maps and CRISPR based gain and loss of function screens
    Cancer Research, 2015
    Co-Authors: Martin Kampmann, Jacqueline E. Villalta, Max A. Horlbeck, Luke A. Gilbert, Britt Adamson, Yuwen Chen, Diego Acostaalvear, Peter Walter, Jonathan S. Weissman
    Abstract:

    Next-generation sequencing has yielded unprecedented insights into the mutational landscapes of cancer cells. The next challenge is to decipher the functional relevance of these mutations for cancer proliferation, metastasis and response to therapy. To tackle these questions, we have developed a versatile functional-genomics platform that overcomes issues that have plagued traditional approaches. Most recently, we have established two novel screening technologies based on the bacterial CRISPR/Cas9 system. In our CRISPRi approach, a catalytically dead version of Cas9 (dCas9) fused to a KRAB transcriptional repressor domain is targeted to specific DNA loci close to the transcription start site of endogenous genes to block transcription. Importantly, and in contrast to RNAi-based approaches, CRISPRi is highly specific and virtually lacks off-target effects. This specificity enabled us to construct more condensed genome-wide libraries. Importantly, CRISPRi proved to be non-toxic and reversible. We also developed an approach for targeted activation of endogenous genes, termed CRISPRa, in which dCas9 recruits an array of transcriptional activators to a transcription start site of interest. CRISPRi-based loss-of-function screens and CRISPRa-based gain-of-function screens yield rich, complementary insights into cellular pathways. In particular, drug resistance in cancer cells is commonly driven by gain-of-function events, which we can model using CRISPRa. In a pilot application, we identify genes whose expression levels control growth and survival of human leukemia cells. While primary genetic screens generate a list of hit genes, it commonly is challenging to elucidate the functional connections between these genes. Systematic, high-density mapping of genetic interactions is a powerful approach to elucidate functional pathways and reveal synthetic lethal / synthetic sick gene pairs, and has successfully been applied in microorganisms. We have developed a functional genomics platform that enables the parallel measurement of 100,000s of double-mutant phenotypes in mammalian cells for the construction of high-density genetic interaction maps. This platform reveals novel biological pathways in cancer cells and points to synthetic-lethal / synthetic-sick gene pairs, which can be exploited therapeutically. It also makes it possible to systematically uncover the accessible escape routes to targeted therapies, paving the way for rational design of combination therapies that pre-empt cancer drug resistance. Systematic comparison of genetic interactions between different growth conditions and genetic backgrounds can reveal context-specific rewiring of functional networks. We present the application of our platforms to identify adaptations and vulnerabilities in the stress response network of leukemia and multiple myeloma cells. Stress response pathways play important roles in cancer cell survival, drug resistance and tumor progression, and cancer cells are particularly dependent on such pathways. A prominent example of such non-oncogene addiction is the hypersensitivity of multiple myeloma cells to proteasome inhibition. We are applying our functional genomics platforms to understand the rewiring of genetic networks in myeloma cells that underlies this non-oncogene addiction and systematically characterize synthetic-lethal vulnerabilities that are potential new targets for combination therapies. Citation Format: Martin Kampmann, Diego Acosta-Alvear, Luke A. Gilbert, Max A. Horlbeck, Min Y. Cho, Britt Adamson, Jacqueline Villalta, Yuwen Chen, Peter Walter, Jonathan S. Weissman. Cancer-specific synthetic lethality and network rewiring elucidated by genetic interaction maps and CRISPR-based gain- and loss-of-function screens. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-56.

  • genome scale CRISPR mediated control of gene repression and activation
    Cell, 2014
    Co-Authors: Luke A. Gilbert, Evan H. Whitehead, Jacqueline E. Villalta, Max A. Horlbeck, Britt Adamson, Yuwen Chen, Barbara Panning, Carla P. Guimaraes, Hidde L. Ploegh, Michael C. Bassik
    Abstract:

    Summary While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

  • CRISPR interference CRISPRi for sequence specific control of gene expression
    Nature Protocols, 2013
    Co-Authors: Luke A. Gilbert, Matthew H. Larson, Jonathan S. Weissman, Xiaowo Wang, Lei S. Qi
    Abstract:

    Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1–2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

  • CRISPR mediated modular rna guided regulation of transcription in eukaryotes
    Cell, 2013
    Co-Authors: Luke A. Gilbert, Leonardo Morsut, Zairan Liu, Sandra E. Torres, Onn Brandman, Evan H. Whitehead, Matthew H. Larson, Noam Sternginossar, Gloria A Brar, Jennifer A Doudna
    Abstract:

    Summary The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

Rodolphe Barrangou - One of the best experts on this subject based on the ideXlab platform.

  • CRISPR-Cas Systems - CRISPR-Cas systems : RNA-mediated adaptive immunity in bacteria and archaea
    2020
    Co-Authors: Rodolphe Barrangou, John Van Der Oost
    Abstract:

    Discovery and seminal developments in the CRISPR field.- Occurrence, diversity of CRISPR-Cas systems and genotyping implications.- Evolution and classification of CRISPR-Cas systems and Cas protein families.- Regulation of CRISPR-based immune responses.- crRNA Biogenesis.- Distribution and mechanism of the Type I CRISPR/Cas Systems.- Type II - Streptococcus thermophilus.- Type III CRISPR-Cas Systems and the Roles CRISPR-Cas in Bacterial Virulence.- CRISPR-Cas Systems to Probe Ecological Diversity and Host-Viral Interactions.- Roles of CRISPR in regulation of physiological processes.- Applications of the versatile CRISPR-Cas systems.- CRISPRs in microbial community context.

  • CRISPR: New Horizons in Phage Resistance and Strain Identification
    Annual Review of Food Science and Technology, 2012
    Co-Authors: Rodolphe Barrangou, Philippe Horvath
    Abstract:

    Bacteria have been widely used as starter cultures in the food industry, notably for the fermentation of milk into dairy products such as cheese and yogurt. Lactic acid bacteria used in food manufacturing, such as lactobacilli, lactococci, streptococci, Leuconostoc, pediococci, and bifidobacteria, are selectively formulated based on functional characteristics that provide idiosyncratic flavor and texture attributes, as well as their ability to withstand processing and manufacturing conditions. Unfortunately, given frequent viral exposure in industrial environments, starter culture selection and development rely on defense systems that provide resistance against bacteriophage predation, including restriction-modification, abortive infection, and recently discovered CRISPRs (clustered regularly interspaced short palindromic repeats). CRISPRs, together with CRISPR-associated genes (cas), form the CRISPR/Cas immune system, which provides adaptive immunity against phages and invasive genetic elements. The immunization process is based on the incorporation of short DNA sequences from virulent phages into the CRISPR locus. Subsequently, CRISPR transcripts are processed into small interfering RNAs that guide a multifunctional protein complex to recognize and cleave matching foreign DNA. Hypervariable CRISPR loci provide insights into the phage and host population dynamics, and new avenues for enhanced phage resistance and genetic typing and tagging of industrial strains.

  • the streptococcus thermophilus CRISPR cas system provides immunity in escherichia coli
    Nucleic Acids Research, 2011
    Co-Authors: Rimantas Sapranauskas, Philippe Horvath, Rodolphe Barrangou, Giedrius Gasiunas, Christophe Fremaux, Virginijus Siksnys
    Abstract:

    The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.

  • the CRISPR cas bacterial immune system cleaves bacteriophage and plasmid dna
    Nature, 2010
    Co-Authors: Josiane E. Garneau, Marie-Ève Dupuis, Manuela Villion, Dennis A Romero, Patrick Boyaval, Philippe Horvath, Alfonso H Magadán, Rodolphe Barrangou, Christophe Fremaux, Sylvain Moineau
    Abstract:

    Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains. CRISPR/Cas is a microbial immune system that is known to protect bacteria from viral infection. It is now shown that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA of both viral and plasmid origin. The system seems remarkably adapted to this end, and it is thought that CRISPR/Cas could be used to naturally generate safer and more robust bacteria that are resistant to the acquisition and spread of antibiotic resistance genes. CRISPR/Cas is a microbial immune system that is known to protect bacteria from virus infection. These authors show that the Streptococcus thermophilus CRISPR/Cas system can prevent both plasmid carriage and phage infection through cleavage of invading double-stranded DNA.

  • The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.
    Nature, 2010
    Co-Authors: Josiane E. Garneau, Marie-Ève Dupuis, Manuela Villion, Dennis A Romero, Patrick Boyaval, Philippe Horvath, Alfonso H Magadán, Rodolphe Barrangou, Christophe Fremaux, Sylvain Moineau
    Abstract:

    Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.

Matthew H. Larson - One of the best experts on this subject based on the ideXlab platform.

  • CRISPR interference CRISPRi for sequence specific control of gene expression
    Nature Protocols, 2013
    Co-Authors: Luke A. Gilbert, Matthew H. Larson, Jonathan S. Weissman, Xiaowo Wang, Lei S. Qi
    Abstract:

    Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1–2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.

  • CRISPR mediated modular rna guided regulation of transcription in eukaryotes
    Cell, 2013
    Co-Authors: Luke A. Gilbert, Leonardo Morsut, Zairan Liu, Sandra E. Torres, Onn Brandman, Evan H. Whitehead, Matthew H. Larson, Noam Sternginossar, Gloria A Brar, Jennifer A Doudna
    Abstract:

    Summary The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells.

  • repurposing CRISPR as an rna guided platform for sequence specific control of gene expression
    Cell, 2013
    Co-Authors: Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jonathan S. Weissman, Adam Paul Arkin, Jennifer A Doudna
    Abstract:

    SUMMARY Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale.

  • Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression
    Cell, 2013
    Co-Authors: Lei S. Qi, Matthew H. Larson, Luke A. Gilbert, Jonathan S. Weissman, Adam Paul Arkin, Jennifer A Doudna, Wendell A. Lim
    Abstract:

    Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-γuided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-γuided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale. © 2013 Elsevier Inc.

  • XCRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes
    Cell, 2013
    Co-Authors: Luke A. Gilbert, Leonardo Morsut, Zairan Liu, Sandra E. Torres, Onn Brandman, Evan H. Whitehead, Noam Stern-ginossar, Matthew H. Larson, Gloria A Brar, Jennifer A Doudna
    Abstract:

    The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells. © 2013 Elsevier Inc.