The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Martin Jinek - One of the best experts on this subject based on the ideXlab platform.
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type iii CRISPR cas systems produce cyclic oligoadenylate second messengers
Nature, 2017Co-Authors: Ole Niewoehner, Carmela Garciadoval, Jakob T Rostol, Christian Berk, Frank Schwede, Laurent Bigler, Jonathan Hall, Luciano A Marraffini, Martin JinekAbstract:In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-Associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-Associated Protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 Proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-Associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
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type iii CRISPR cas systems generate cyclic oligoadenylate second messengers to activate csm6 rnases
bioRxiv, 2017Co-Authors: Ole Niewoehner, Carmela Garciadoval, Jakob T Rostol, Christian Berk, Frank Schwede, Laurent Bigler, Jonathan Hall, Luciano A Marraffini, Martin JinekAbstract:In many prokaryotes, type III CRISPR-Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-Associated Protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 Proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
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structural basis for guide rna processing and seed dependent dna targeting by CRISPR cas12a
Molecular Cell, 2017Co-Authors: Daan C Swarts, John Van Der Oost, Martin JinekAbstract:Summary The CRISPR-Associated Protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies.
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structural basis for the endoribonuclease activity of the type iii a CRISPR Associated Protein csm6
RNA, 2016Co-Authors: Ole Niewoehner, Martin JinekAbstract:Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-Associated Protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the Protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 Proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.
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structural basis of pam dependent target dna recognition by the cas9 endonuclease
Nature, 2014Co-Authors: Carolin Anders, Ole Niewoehner, Alessia Duerst, Martin JinekAbstract:The CRISPR-Associated Protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.
Jin-soo Kim - One of the best experts on this subject based on the ideXlab platform.
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Genome editing comes of age
NATURE PUBLISHING GROUP, 2018Co-Authors: Jin-soo KimAbstract:Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)–Cas9 (CRISPR-Associated Protein 9) RNA-guided endonucleases (RGENs). I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR–Cas9 genome editing approaches that has revolutionized the field. © 2016 Nature America, Inc. All rights reserved.121
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Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY, 2018Co-Authors: Taeyoung Koo, Jungjoon Lee, Jin-soo KimAbstract:Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid offtarget mutations. (c) The Korean Society for Molecular and Cellular Biology. All rights reserved.556
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Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
NATURE PUBLISHING GROUP, 2018Co-Authors: Toungbin Lim, Jin-soo Kim, So Young Bak, Keewon Sung, Euihwan Jeong, Seung Hwan Lee, Sangsu Bae, Seong Keun KimAbstract:The type II CRISPR-Associated Protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system. © The Author(s) 2016181
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selective disruption of an oncogenic mutant allele by CRISPR cas9 induces efficient tumor regression
Nucleic Acids Research, 2017Co-Authors: Taeyoung Koo, Sangsu Bae, Arum Yoon, Heeyeon Cho, Chaeok Yun, Jin-soo KimAbstract:Approximately 15% of non-small cell lung cancer cases are Associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-Associated diseases.
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measuring and reducing off target activities of programmable nucleases including CRISPR cas9
Molecules and Cells, 2015Co-Authors: Taeyoung Koo, Jungjoon K Lee, Jin-soo KimAbstract:Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.
Ole Niewoehner - One of the best experts on this subject based on the ideXlab platform.
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type iii CRISPR cas systems produce cyclic oligoadenylate second messengers
Nature, 2017Co-Authors: Ole Niewoehner, Carmela Garciadoval, Jakob T Rostol, Christian Berk, Frank Schwede, Laurent Bigler, Jonathan Hall, Luciano A Marraffini, Martin JinekAbstract:In many prokaryotes, type III clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-Associated (Cas) systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex. The CRISPR-Associated Protein Csm6 additionally contributes to interference by functioning as a standalone RNase that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 Proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the interference complex, its Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CRISPR-Associated Rossmann fold (CARF) domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to an unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
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type iii CRISPR cas systems generate cyclic oligoadenylate second messengers to activate csm6 rnases
bioRxiv, 2017Co-Authors: Ole Niewoehner, Carmela Garciadoval, Jakob T Rostol, Christian Berk, Frank Schwede, Laurent Bigler, Jonathan Hall, Luciano A Marraffini, Martin JinekAbstract:In many prokaryotes, type III CRISPR-Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-Associated Protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 Proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.
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structural basis for the endoribonuclease activity of the type iii a CRISPR Associated Protein csm6
RNA, 2016Co-Authors: Ole Niewoehner, Martin JinekAbstract:Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-Associated Protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the Protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 Proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.
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structural basis of pam dependent target dna recognition by the cas9 endonuclease
Nature, 2014Co-Authors: Carolin Anders, Ole Niewoehner, Alessia Duerst, Martin JinekAbstract:The CRISPR-Associated Protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.
Taeyoung Koo - One of the best experts on this subject based on the ideXlab platform.
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Measuring and Reducing Off-Target Activities of Programmable Nucleases Including CRISPR-Cas9
KOREAN SOC MOLECULAR & CELLULAR BIOLOGY, 2018Co-Authors: Taeyoung Koo, Jungjoon Lee, Jin-soo KimAbstract:Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid offtarget mutations. (c) The Korean Society for Molecular and Cellular Biology. All rights reserved.556
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selective disruption of an oncogenic mutant allele by CRISPR cas9 induces efficient tumor regression
Nucleic Acids Research, 2017Co-Authors: Taeyoung Koo, Sangsu Bae, Arum Yoon, Heeyeon Cho, Chaeok Yun, Jin-soo KimAbstract:Approximately 15% of non-small cell lung cancer cases are Associated with a mutation in the epidermal growth factor receptor (EGFR) gene, which plays a critical role in tumor progression. With the goal of treating mutated EGFR-mediated lung cancer, we demonstrate the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR Associated Protein 9 (Cas9) system to discriminate between the oncogenic mutant and wild-type EGFR alleles and eliminate the carcinogenic mutant EGFR allele with high accuracy. We targeted an EGFR oncogene harboring a single-nucleotide missense mutation (CTG > CGG) that generates a protospacer-adjacent motif sequence recognized by the CRISPR/Cas9 derived from Streptococcus pyogenes. Co-delivery of Cas9 and an EGFR mutation-specific single-guide RNA via adenovirus resulted in precise disruption at the oncogenic mutation site with high specificity. Furthermore, this CRISPR/Cas9-mediated mutant allele disruption led to significantly enhanced cancer cell killing and reduced tumor size in a xenograft mouse model of human lung cancer. Taken together, these results indicate that targeting an oncogenic mutation using CRISPR/Cas9 offers a powerful surgical strategy to disrupt oncogenic mutations to treat cancers; similar strategies could be used to treat other mutation-Associated diseases.
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measuring and reducing off target activities of programmable nucleases including CRISPR cas9
Molecules and Cells, 2015Co-Authors: Taeyoung Koo, Jungjoon K Lee, Jin-soo KimAbstract:Programmable nucleases, which include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) repurposed from the type II clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) system are now widely used for genome editing in higher eukaryotic cells and whole organisms, revolutionising almost every discipline in biological research, medicine, and biotechnology. All of these nucleases, however, induce off-target mutations at sites homologous in sequence with on-target sites, limiting their utility in many applications including gene or cell therapy. In this review, we compare methods for detecting nuclease off-target mutations. We also review methods for profiling genome-wide off-target effects and discuss how to reduce or avoid off-target mutations.
Sílvia Coimbra - One of the best experts on this subject based on the ideXlab platform.
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The best CRISPR/Cas9 versus RNA interference approaches for Arabinogalactan Proteins’ study
Molecular Biology Reports, 2020Co-Authors: Diana Moreira, Ana Marta Pereira, Ana Lúcia Lopes, Sílvia CoimbraAbstract:Arabinogalactan Proteins (AGPs) are hydroxyproline-rich Proteins containing a high proportion of carbohydrates, widely spread in the plant kingdom. AGPs have been suggested to play important roles in plant development processes, especially in sexual plant reproduction. Nevertheless, the functions of a large number of these molecules, remains to be discovered. In this review, we discuss two revolutionary genetic techniques that are able to decode the roles of these glycoProteins in an easy and efficient way. The RNA interference is a frequently technique used in plant biology that promotes genes silencing. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—Associated Protein 9 (CRISPR/Cas9), emerged a few years ago as a revolutionary genome-editing technique that has allowed null mutants to be obtained in a wide variety of organisms, including plants. The two techniques have some differences between them and depending on the research objective, these may work as advantage or disadvantage. In the present work, we propose the use of the two techniques to obtain AGP mutants easily and quickly, helping to unravel the role of AGPs, surely a great asset for the future.
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the best CRISPR cas9 versus rna interference approaches for arabinogalactan Proteins study
Molecular Biology Reports, 2020Co-Authors: Diana Moreira, Ana Marta Pereira, Ana Lúcia Lopes, Sílvia CoimbraAbstract:Arabinogalactan Proteins (AGPs) are hydroxyproline-rich Proteins containing a high proportion of carbohydrates, widely spread in the plant kingdom. AGPs have been suggested to play important roles in plant development processes, especially in sexual plant reproduction. Nevertheless, the functions of a large number of these molecules, remains to be discovered. In this review, we discuss two revolutionary genetic techniques that are able to decode the roles of these glycoProteins in an easy and efficient way. The RNA interference is a frequently technique used in plant biology that promotes genes silencing. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—Associated Protein 9 (CRISPR/Cas9), emerged a few years ago as a revolutionary genome-editing technique that has allowed null mutants to be obtained in a wide variety of organisms, including plants. The two techniques have some differences between them and depending on the research objective, these may work as advantage or disadvantage. In the present work, we propose the use of the two techniques to obtain AGP mutants easily and quickly, helping to unravel the role of AGPs, surely a great asset for the future.