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Stan J. J. Brouns - One of the best experts on this subject based on the ideXlab platform.
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interference driven spacer acquisition is dominant over naive and primed adaptation in a native crispr cas system
Nature Communications, 2016Co-Authors: Raymond H.j. Staals, Ambarish Biswas, Chris M Brown, Simon A. Jackson, Stan J. J. Brouns, Peter C FineranAbstract:CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3'-5' translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition ('targeted acquisition') is a major contributor to adaptation in type I-F CRISPR-Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
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interference driven spacer acquisition is dominant over naive and primed adaptation in a native crispr cas system
Nature Communications, 2016Co-Authors: Raymond H.j. Staals, Ambarish Biswas, Chris M Brown, Simon A. Jackson, Stan J. J. Brouns, Peter C FineranAbstract:CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition’) is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome. Prokaryotic CRISPR-Cas systems provide adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, Staals et al. analyse millions of such events in a native CRISPR-Cas system, showing that newly acquired spacers provoke additional rounds of spacer acquisition.
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crispr interference directs strand specific spacer acquisition
PLOS ONE, 2012Co-Authors: Daan C Swarts, Cas Mosterd, Mark Wj Van Passel, Stan J. J. BrounsAbstract:Background CRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference. Results We describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner. Conclusions The E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster – in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.
Kimberley D Seed - One of the best experts on this subject based on the ideXlab platform.
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competition between mobile genetic elements drives optimization of a phage encoded crispr cas system insights from a natural arms race
Philosophical Transactions of the Royal Society B, 2019Co-Authors: Amelia C Mckitterick, Kristen N Legault, Angus Angermeyer, Munirul Alam, Kimberley D SeedAbstract:CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defence against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not interfere equally with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and is dependent on Cas2-3 helicase activity. We propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory-acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.
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competition between mobile genetic elements drives optimization of a phage encoded crispr cas system insights from a natural arms race
bioRxiv, 2018Co-Authors: Amelia C Mckitterick, Kristen N Legault, Angus Angermeyer, Munirul Alam, Kimberley D SeedAbstract:CRISPR-Cas systems function as adaptive immune systems by acquiring nucleotide sequences called spacers that mediate sequence-specific defense against competitors. Uniquely, the phage ICP1 encodes a Type I-F CRISPR-Cas system that is deployed to target and overcome PLE, a mobile genetic element with anti-phage activity in Vibrio cholerae. Here, we exploit the arms race between ICP1 and PLE to examine spacer acquisition and interference under laboratory conditions to reconcile findings from wild populations. Natural ICP1 isolates encode multiple spacers directed against PLE, but we find that single spacers do not equally interfere with PLE mobilization. High-throughput sequencing to assay spacer acquisition reveals that ICP1 can also acquire spacers that target the V. cholerae chromosome. We find that targeting the V. cholerae chromosome proximal to PLE is sufficient to block PLE and propose a model in which indirect chromosomal spacers are able to circumvent PLE by Cas2-3-mediated processive degradation of the V. cholerae chromosome before PLE mobilization. Generally, laboratory acquired spacers are much more diverse than the subset of spacers maintained by ICP1 in nature, showing how evolutionary pressures can constrain CRISPR-Cas targeting in ways that are often not appreciated through in vitro analyses.
Peter C Fineran - One of the best experts on this subject based on the ideXlab platform.
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interference driven spacer acquisition is dominant over naive and primed adaptation in a native crispr cas system
Nature Communications, 2016Co-Authors: Raymond H.j. Staals, Ambarish Biswas, Chris M Brown, Simon A. Jackson, Stan J. J. Brouns, Peter C FineranAbstract:CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition’) is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome. Prokaryotic CRISPR-Cas systems provide adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, Staals et al. analyse millions of such events in a native CRISPR-Cas system, showing that newly acquired spacers provoke additional rounds of spacer acquisition.
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interference driven spacer acquisition is dominant over naive and primed adaptation in a native crispr cas system
Nature Communications, 2016Co-Authors: Raymond H.j. Staals, Ambarish Biswas, Chris M Brown, Simon A. Jackson, Stan J. J. Brouns, Peter C FineranAbstract:CRISPR-Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR-Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR-Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3'-5' translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition ('targeted acquisition') is a major contributor to adaptation in type I-F CRISPR-Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome.
C Ocallaghan - One of the best experts on this subject based on the ideXlab platform.
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research articlesinvestigating improving powder deagglomeration via dry powder inhalers at a low inspiratory flow rate by employing add on spacers
Journal of Pharmaceutical Sciences, 2008Co-Authors: Touraj Ehtezazi, D R Allanson, Ian Jenkinson, Ian Shrubb, C OcallaghanAbstract:The aim of this study was to investigate whether small add‐on spacers alone or equipped with a passive deagglomerating component would improve aerosol performances of passive low airflow resistance dry powder inhalers (DPIs) at a low inhalation flow rate. Depositions of beclometasone dipropionate (BDP) and salbutamol sulphate (SS) via the Asmabec Clickhaler and Asmasal Clickhaler at 30 L/min airflow rate in an oropharyngeal model and attached filter were determined. Three add‐on spacers, one with 5.0 cm length, and the other with the same features but incorporating a fine mesh, and the third one with the length of 8.5 cm (long add‐on spacer) were used. Incorporating mesh did not improve the filter dose for SS, and significantly reduced this dose for BDP. The long add‐on spacer was the most efficient spacer as it had minimal effects on the filter doses, also significantly reduced drug depositions in the model. In conclusion, an optimum length of an add‐on spacer is required to minimise oropharyngeal drug deposition via a low airflow resistance DPI at a low inspiratory flow rate without considerable reduction of the respirable dose. Incorporating sieves within add‐on spacers may diminish aerosol performances of the DPIs at low airflow rates. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5212–5221, 2008
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investigating improving powder deagglomeration via dry powder inhalers at a low inspiratory flow rate by employing add on spacers
Journal of Pharmaceutical Sciences, 2008Co-Authors: Touraj Ehtezazi, D R Allanson, Ian Jenkinson, Ian Shrubb, C OcallaghanAbstract:Abstract The aim of this study was to investigate whether small add‐on spacers alone or equipped with a passive deagglomerating component would improve aerosol performances of passive low airflow resistance dry powder inhalers (DPIs) at a low inhalation flow rate. Depositions of beclometasone dipropionate (BDP) and salbutamol sulphate (SS) via the Asmabec Clickhaler and Asmasal Clickhaler at 30 L/min airflow rate in an oropharyngeal model and attached filter were determined. Three add‐on spacers, one with 5.0 cm length, and the other with the same features but incorporating a fine mesh, and the third one with the length of 8.5 cm (long add‐on spacer) were used. Incorporating mesh did not improve the filter dose for SS, and significantly reduced this dose for BDP. The long add‐on spacer was the most efficient spacer as it had minimal effects on the filter doses, also significantly reduced drug depositions in the model. In conclusion, an optimum length of an add‐on spacer is required to minimise oropharyngeal drug deposition via a low airflow resistance DPI at a low inspiratory flow rate without considerable reduction of the respirable dose. Incorporating sieves within add‐on spacers may diminish aerosol performances of the DPIs at low airflow rates. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5212–5221, 2008
Thomas Reith - One of the best experts on this subject based on the ideXlab platform.
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novel spacers for mass transfer enhancement in membrane separations
Journal of Membrane Science, 2005Co-Authors: Wytze Meindersma, A.b. De Haan, Thomas ReithAbstract:The optimal flow pattern for mass transfer enhancement in spacer-filled channels is characterized by the coexistence of transversal and longitudinal vortices in the flow close to the channel walls and minimal cross-flow power consumption in the middle of the channel. The mass transfer enhancement of spacers with modified filaments, twisted tapes and multi-layer structures, which were expected to generate these flow patterns, was investigated experimentally. The results indicate that the performance of spacers with modified filaments and twisted tapes is generally worse while the performance of spacers with multi-layer structure is generally better than that of the optimal non-woven net spacer. An optimal multi-layer spacer was designed with optimal non-woven nets in the outer layers and twisted tapes in the middle-layer. Its average Sherwood number is about 30% higher than the Sherwood number of the optimal non-woven spacer at the same cross-flow power consumption whereas the cross-flow power consumption is only about 40% of the consumption of the optimal non-woven spacer at the same Sherwood number. The Reynolds number based on the height of a spacer-filled channel varies from 40 to 500 in present study.
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optimization of commercial net spacers in spiral wound membrane modules
Journal of Membrane Science, 2002Co-Authors: Wytze Meindersma, A.b. De Haan, Thomas ReithAbstract:CFD simulations have been used to determine mass transfer coefficients and power consumption of commercial net spacers. The simulations show transversal and longitudinal vortices, vortex shedding and instationary flow behavior leading to the enhanced mass transfer in spacer filled-channels compared to empty channels. The results of the simulations were validated with experiments and compared with data reported in literature, showing satisfactory agreement. Furthermore, CFD simulations were used to optimize the geometry of commercial net spacers in terms of mass transfer and power consumption. The performance of these optimized spacer geometries will be used as reference for future work on the development of new high-performance spacer shapes.
