The Experts below are selected from a list of 1839 Experts worldwide ranked by ideXlab platform
Olga Musharova - One of the best experts on this subject based on the ideXlab platform.
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systematic analysis of type i e escherichia coli CRISPR cas pam sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina SemenovaAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Stan J J Brouns, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina Semenova, Konstantin SeverinovAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
Nucleic Acids Research, 2017Co-Authors: Olga Musharova, Konstantin Severinov, Kirill A Datsenko, Ekaterina Semenova, Evgeny Klimuk, Anastasia Metlitskaya, Maria D. Logacheva, Ekaterina SavitskayaAbstract:During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR Interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR Interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
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CRISPR Interference and priming varies with individual spacer sequences
Nucleic Acids Research, 2015Co-Authors: Arun S Seetharam, Stan J J Brouns, Olga Musharova, Konstantin Severinov, Andrew J Severin, Dipali G SashitalAbstract:CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR Interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block Interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for Interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR Interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote Interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naive' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for Interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.
Stan J J Brouns - One of the best experts on this subject based on the ideXlab platform.
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Direct Visualization of Native CRISPR Target Search in Live Bacteria Reveals Cascade DNA Surveillance Mechanism.
Molecular Cell, 2019Co-Authors: Jochem N. A. Vink, Marnix Vlot, Cristóbal Almendros, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Stan J J BrounsAbstract:Summary CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR Interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR Interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.
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Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Stan J J Brouns, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina Semenova, Konstantin SeverinovAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Direct visualization of native CRISPR target search in live bacteria reveals Cascade DNA surveillance mechanism
bioRxiv, 2019Co-Authors: Jochem N. A. Vink, Marnix Vlot, Cristóbal Almendros, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Stan J J BrounsAbstract:Abstract CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR Interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR Interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment. One sentence summary The results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.
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Role of nucleotide identity in effective CRISPR target escape mutations
Nucleic Acids Research, 2018Co-Authors: Tim Künne, Rebecca E. Mckenzie, Fausia Da Silva, Nico Konstantinides, Ryan N. Jackson, Stan J J BrounsAbstract:Prokaryotes use primed CRISPR adaptation to update their memory bank of spacers against invading genetic elements that have escaped CRISPR Interference through mutations in their protospacer target site. We previously observed a trend that nucleotide-dependent mismatches between crRNA and the protospacer strongly influence the efficiency of primed CRISPR adaptation. Here we show that guanine-substitutions in the target strand of the protospacer are highly detrimental to CRISPR Interference and Interference-dependent priming, while cytosine-substitutions are more readily tolerated. Furthermore, we show that this effect is based on strongly decreased binding affinity of the effector complex Cascade for guanine-mismatched targets, while cytosine-mismatched targets only minimally affect target DNA binding. Structural modeling of Cascade-bound targets with mismatches shows that steric clashes of mismatched guanines lead to unfavorable conformations of the RNA-DNA duplex. This effect has strong implications for the natural selection of target site mutations that lead to effective escape from type I CRISPR-Cas systems.
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Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation
Cell Reports, 2018Co-Authors: Sebastian N. Kieper, Cristóbal Almendros, Juliane Behler, Rebecca E. Mckenzie, Franklin Luzia Nóbrega, Anna C. Haagsma, Jochem N. A. Vink, Wolfgang R. Hess, Stan J J BrounsAbstract:Summary CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1 , cas2 , and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR Interference in the native host Synechocystis . We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR Interference, providing a better chance of surviving infection by mobile genetic elements.
Ekaterina Semenova - One of the best experts on this subject based on the ideXlab platform.
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Rapid Multiplex Creation of Escherichia coli Strains Capable of Interfering with Phage Infection Through CRISPR.
Methods of Molecular Biology, 2020Co-Authors: Alexandra Strotksaya, Ekaterina Semenova, Ekaterina Savitskaya, Konstantin SeverinovAbstract:In Escherichia coli, acquisition of new spacers in the course of CRISPR-Cas adaptation is dramatically stimulated by preexisting partial matches between bacterial CRISPR cassette spacer and a protospacer sequence in DNA of infecting bacteriophage or plasmid transformed. This phenomenon, which we refer to as “priming”, can be used for very simple and rapid construction of multiple E. coli strains capable of targeting, through CRISPR Interference, any phage or plasmid of interest. Availability of such strains should allow rapid progress in the analysis of CRISPR-Cas system function against diverse mobile genetic elements.
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systematic analysis of type i e escherichia coli CRISPR cas pam sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina SemenovaAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Stan J J Brouns, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina Semenova, Konstantin SeverinovAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
Nucleic Acids Research, 2017Co-Authors: Olga Musharova, Konstantin Severinov, Kirill A Datsenko, Ekaterina Semenova, Evgeny Klimuk, Anastasia Metlitskaya, Maria D. Logacheva, Ekaterina SavitskayaAbstract:During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR Interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR Interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.
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The Influence of Copy-Number of Targeted Extrachromosomal Genetic Elements on the Outcome of CRISPR-Cas Defense
Frontiers in Molecular Biosciences, 2016Co-Authors: Konstantin Severinov, Iaroslav Ispolatov, Ekaterina SemenovaAbstract:Prokaryotic type I CRISPR-Cas systems respond to the presence of mobile genetic elements such as plasmids and phages in two different ways. CRISPR Interference efficiently destroys foreign DNA harbouring protospacers fully matching CRISPR RNA spacers. In contrast, even a single mismatch between a spacer and a protospacer can render CRISPR Interference ineffective but causes primed adaptation - efficient and specific acquisition of additional spacers from foreign DNA into the CRISPR array of the host. It has been proposed that the Interference and primed adaptation pathways are mediated by structurally different complexes formed by the effector Cascade complex on matching and mismatched protospacers. Here, we present experimental evidence and present a simple mathematical model that shows that when plasmid copy number maintenance/phage genome replication is taken into account, the two apparently different outcomes of the CRISPR-Cas response can be accounted for by just one kind of effector complex on both targets. The results underscore the importance of consideration of targeted genome biology when considering consequences of CRISPR-Cas systems action.
Dipali G Sashital - One of the best experts on this subject based on the ideXlab platform.
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fluorescence based methods for measuring target Interference by CRISPR cas systems
Methods in Enzymology, 2019Co-Authors: Phong T Phan, Michael A Schelling, Dipali G SashitalAbstract:Abstract Type I, II, and V CRISPR–Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR Interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR Interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Given the importance of Cas effectors in host defense and for biotechnology tools, there has been considerable interest in developing sensitive methods for detecting Cas effector activity through CRISPR Interference. In this chapter, we describe an in vivo fluorescence-based method for monitoring plasmid Interference in Escherichia coli. This approach uses a green fluorescent protein reporter to monitor varying plasmid levels within bacterial colonies, or to measure the rate of plasmid-loss in bacterial populations over time. We demonstrate the use of this simple plasmid-loss assay for both chromosomally integrated and plasmid-borne CRISPR–Cas systems.
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CRISPR Interference and priming varies with individual spacer sequences
Nucleic Acids Research, 2015Co-Authors: Arun S Seetharam, Stan J J Brouns, Olga Musharova, Konstantin Severinov, Andrew J Severin, Dipali G SashitalAbstract:CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems allow bacteria to adapt to infection by acquiring 'spacer' sequences from invader DNA into genomic CRISPR loci. Cas proteins use RNAs derived from these loci to target cognate sequences for destruction through CRISPR Interference. Mutations in the protospacer adjacent motif (PAM) and seed regions block Interference but promote rapid 'primed' adaptation. Here, we use multiple spacer sequences to reexamine the PAM and seed sequence requirements for Interference and priming in the Escherichia coli Type I-E CRISPR-Cas system. Surprisingly, CRISPR Interference is far more tolerant of mutations in the seed and the PAM than previously reported, and this mutational tolerance, as well as priming activity, is highly dependent on spacer sequence. We identify a large number of functional PAMs that can promote Interference, priming or both activities, depending on the associated spacer sequence. Functional PAMs are preferentially acquired during unprimed 'naive' adaptation, leading to a rapid priming response following infection. Our results provide numerous insights into the importance of both spacer and target sequences for Interference and priming, and reveal that priming is a major pathway for adaptation during initial infection.
Ekaterina Savitskaya - One of the best experts on this subject based on the ideXlab platform.
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Rapid Multiplex Creation of Escherichia coli Strains Capable of Interfering with Phage Infection Through CRISPR.
Methods of Molecular Biology, 2020Co-Authors: Alexandra Strotksaya, Ekaterina Semenova, Ekaterina Savitskaya, Konstantin SeverinovAbstract:In Escherichia coli, acquisition of new spacers in the course of CRISPR-Cas adaptation is dramatically stimulated by preexisting partial matches between bacterial CRISPR cassette spacer and a protospacer sequence in DNA of infecting bacteriophage or plasmid transformed. This phenomenon, which we refer to as “priming”, can be used for very simple and rapid construction of multiple E. coli strains capable of targeting, through CRISPR Interference, any phage or plasmid of interest. Availability of such strains should allow rapid progress in the analysis of CRISPR-Cas system function against diverse mobile genetic elements.
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systematic analysis of type i e escherichia coli CRISPR cas pam sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina SemenovaAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Systematic analysis of Type I‐E Escherichia coli CRISPR‐Cas PAM sequences ability to promote Interference and primed adaptation
Molecular Microbiology, 2019Co-Authors: Olga Musharova, Stan J J Brouns, Vasily Sitnik, Marnix Vlot, Ekaterina Savitskaya, Kirill A Datsenko, Andrey Krivoy, Ivan Fedorov, Ekaterina Semenova, Konstantin SeverinovAbstract:CRISPR Interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» – acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient Interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR Interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause Interference, those that support rapid Interference and those that lead to reduced Interference that occurs over extended periods of time. PAM variants unable to support Interference also did not support primed adaptation; those that supported rapid Interference led to no or low levels of adaptation, while those that caused attenuated levels of Interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR Interference. Extended over time Interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition.
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Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation
Nucleic Acids Research, 2017Co-Authors: Olga Musharova, Konstantin Severinov, Kirill A Datsenko, Ekaterina Semenova, Evgeny Klimuk, Anastasia Metlitskaya, Maria D. Logacheva, Ekaterina SavitskayaAbstract:During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR Interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR Interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array.