The Experts below are selected from a list of 60081 Experts worldwide ranked by ideXlab platform
Jennifer A Doudna - One of the best experts on this subject based on the ideXlab platform.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
Matias Kaplan - One of the best experts on this subject based on the ideXlab platform.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
Mitchell R Oconnell - One of the best experts on this subject based on the ideXlab platform.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
Ari Sihvola - One of the best experts on this subject based on the ideXlab platform.
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resonant scattering characteristics of homogeneous dielectric sphere
IEEE Transactions on Antennas and Propagation, 2017Co-Authors: Dimitrios C. Tzarouchis, Pasi Ylaoijala, Ari SihvolaAbstract:In this paper, the classical problem of electromagnetic scattering by a single homogeneous sphere is revisited. Main focus is the study of the scattering behavior as a function of the material contrast and the size parameters for all electric and magnetic resonances of a dielectric sphere. Specifically, the Pade approximants are introduced and utilized as an alternative system expansion of the Mie coefficients. Low order Pade approximants can give compact and physically insightful expressions for the scattering system and the enabled dynamic mechanisms. Higher order approximants are used for predicting accurately the resonant pole spectrum. These results are summarized into general pole formulae, covering up to fifth order magnetic and forth order electric resonances of a small dielectric sphere. Additionally, the Connection between the radiative damping process and the resonant linewidth is investigated. The results obtained reveal the Fundamental Connection of the radiative damping mechanism with the maximum width occurring for each resonance. Finally, the suggested system ansatz is used for studying the resonant absorption maximum through a circuit-inspired perspective.
Alexandra Eastseletsky - One of the best experts on this subject based on the ideXlab platform.
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programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:In the presence of a short DNA oligonucleotide containing a protospacer adjacent motif, a guide-RNA-programmed Cas9 is able to specifically bind and/or cleave single-stranded RNA—this system can be used to isolate specific endogenous RNA transcripts from a cell lysate without any tag or modification. The bacterial CRISPR immune defence system, and its effector Cas9 in particular, have recently been exploited for sequence-specific genome editing in eukaryotic cells. Cas9 binds a guide RNA and in the presence of a DNA motif known as protospacer adjacent motif (PAM), is able to cleave the target DNA. New work by Jennifer Doudna and colleagues reveals the unexpected result that in the presence of a DNA oligomer containing PAM, a guide RNA-programmed Cas9 is able to cleave single-stranded RNA as well. They show that this system can also be used to isolate specific endogenous RNA transcripts, without any tag or modification, from a cell lysate. Thus, the system can be programmed to either bind or cut desired RNA targets, depending on the PAM used. This work and points the way towards possible new technologies for programmable RNA recognition. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1,2,3,4,5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4,5,6,7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
-
programmable rna recognition and cleavage by crispr cas9
Nature, 2014Co-Authors: Mitchell R Oconnell, Benjamin L Oakes, Samuel H Sternberg, Alexandra Eastseletsky, Matias Kaplan, Jennifer A DoudnaAbstract:The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a Fundamental Connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
