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Dinshaw J Patel - One of the best experts on this subject based on the ideXlab platform.
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accommodation of helical imperfections in rhodobacter sphaeroides argonaute teRNAry complexes with Guide RNA and target dna
Cell Reports, 2018Co-Authors: Daria Esyunina, Ivan Olovnikov, M Teplova, Andrey Kulbachinskiy, Alexei A Aravin, Dinshaw J PatelAbstract:Prokaryotic Argonaute (Ago) proteins were recently shown to target foreign genetic elements, thus making them a perfect model for studies of interference mechanisms. Here, we study interactions of Rhodobacter sphaeroides Ago (RsAgo) with Guide RNA (gRNA) and fully complementary or imperfect target DNA (tDNA) using biochemical and structural approaches. We show that RsAgo can specifically recognize both the first nucleotide in gRNA and complementary nucleotide in tDNA, and both interactions contribute to nucleic acid binding. Non-canonical pairs and bulges on the target strand can be accommodated by RsAgo with minimal perturbation of the duplex but significantly reduce RsAgo affinity to tDNA. Surprisingly, mismatches between gRNA and tDNA induce dissociation of the Guide-target duplex from RsAgo. Our results reveal plasticity in the ability of Ago proteins to accommodate helical imperfections, show how this might affect the efficiency of RNA silencing, and suggest a potential mechanism for Guide release and Ago recycling.
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structure of yeast argonaute with Guide RNA
Nature, 2012Co-Authors: Kotaro Nakanishi, David E Weinberg, David P Bartel, Dinshaw J PatelAbstract:The RNA-induced silencing complex, comprising Argonaute and Guide RNA, mediates RNA interference. Here we report the 3.2 A crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with Guide RNA originating from small-RNA duplexes autonomously loaded and processed by recombinant KpAGO. Despite their diverse sequences, Guide-RNA nucleotides 1–8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2′-hydroxyl groups pre-organizing the backbone of nucleotides 2–8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of Guide–target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage. Argonaute proteins are an essential part of the Guide-RNA–protein complex that carries out RNA-induced gene silencing; structure–function studies of the yeast complex reveal conserved features of the eukaryotic complex, which underlie formation of the catalytically active conformation. The functional complex that carries out RNA-induced gene silencing consists of an Argonaute (Ago) protein bound to a short single-stranded Guide RNA. This complex recognizes and binds a complementary messenger RNA sequence and mediates either RNA cleavage or repression of its translation. The 3.2-Angstrom crystal structure of Kluyveromyces polysporus Argonaute, bound by chance to nonspecific Guide RNA, has now been determined. The prokaryotic and human Ago protein structures have been solved in inactive conformations, but this yeast Ago structure is in an active conformation that reveals the mechanism of catalysis.
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structural basis for 5 end specific recognition of Guide RNA by the a fulgidus piwi protein
Nature, 2005Co-Authors: Yuren Yuan, Gunter Meister, Thomas Tuschl, Dinshaw J PatelAbstract:RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism1,2,3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded Guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNAse H fold4,5 critical for the endonuclease cleavage activity of RISC4,5,6. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for Guide-strand 5′-end recognition and providing insight into Guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the Guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the Guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi–RNA complex, and that determined elsewhere7, provide direct support for the 5′ region of the Guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the Guide RNA.
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Structural basis for 5′-end-specific recognition of Guide RNA by the A. fulgidus Piwi protein
Nature, 2005Co-Authors: Jin-biao Ma, Gunter Meister, Yuren Yuan, Thomas Tuschl, Dinshaw J PatelAbstract:RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism1,2,3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded Guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNAse H fold4,5 critical for the endonuclease cleavage activity of RISC4,5,6. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for Guide-strand 5′-end recognition and providing insight into Guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the Guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the Guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi–RNA complex, and that determined elsewhere7, provide direct support for the 5′ region of the Guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the Guide RNA.
Martin Jinek - One of the best experts on this subject based on the ideXlab platform.
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Preparation and electroporation of Cas12a/Cpf1-Guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells.
Methods in Enzymology, 2019Co-Authors: Lucas Kissling, Daan C Swarts, Asun Monfort, Anton Wutz, Martin JinekAbstract:Abstract CRISPR–Cas12a is a bacterial RNA-Guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-Guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of Guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.
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Structural basis for Guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
Molecular Cell, 2017Co-Authors: Daan C Swarts, Martin Jinek, John Van Der OostAbstract: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 in a binary complex with a Guide RNA, and in a R-loop complex containing a non-cleavable Guide RNA precursor and full-length target DNA. Corroborated by biochemical experiments, these structures elucidate the mechanisms of Guide RNA processing and pre-ordering of the seed sequence in the Guide RNA that primes Cas12a for target DNA binding. The R-loop complex structure furthermore 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 that may contribute to further development of genome editing technologies.
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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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In Vitro Reconstitution and Crystallization of Cas9 Endonuclease Bound to a Guide RNA and a DNA Target.
Methods in Enzymology, 2015Co-Authors: C. Anders, Ole Niewoehner, Martin JinekAbstract:The programmable RNA-Guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA Guide or single-molecule Guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the Guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-Guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a teRNAry complex with a Guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.
Niren Murthy - One of the best experts on this subject based on the ideXlab platform.
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a cleavage responsive stem loop hairpin for assaying Guide RNA activity
ACS Chemical Biology, 2018Co-Authors: Tara R Deboer, Noreen Wauford, Jingyi Chung, Miguel Perez, Niren MurthyAbstract:The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the Guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alteRNAtive Guide RNA validation system called GuideR. GuideR features a hairpin loop structure with a proximal guanosine-rich unit, a distal fluorophore unit, and a gRNA-targeting stem component. Cleavage of GuideR by its complementary RNA-Guided Cas9 endonuclease complex yields a fluorescent emission at 525 nm, signaling effective cleavage of the hairpin structure. GuideR was validated using the model gene target mpcsk9, and it was able to identify the gRNA that could most efficiently cleave the target mpcsk9 gene. The modular design of GuideR should allow it to have broad applicability in validating gRNAs, and its fluorescent signal output of...
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synthetically modified Guide RNA and donor dna are a versatile platform for crispr cas9 engineering
eLife, 2017Co-Authors: Vanessa A Mackley, Anthony T Chong, Mark A Dewitt, Jacob E Corn, Niren MurthyAbstract:There are several different technologies that can be used to make specific changes to particular genes in cells. These “gene editing” approaches have the potential to help humans in a variety of different ways, for example, to treat diseases that presently have no cure, or to improve the nutritional quality of crop plants. One such gene editing approach is known as CRISPR. To edit a specific gene, a molecule called a Guide ribonucleic acid (or Guide RNA for short) binds to a section of the gene and recruits an enzyme to cut the DNA encoding the gene in a particular location. Adding a “donor” DNA molecule that contains the desired “edit” can lead to the cell repairing the broken gene in a way that incorporates the desired change. Modifying the Guide RNA or the donor DNA can enhance CRISPR editing. For example, extending the Guide RNA molecules by adding “aptamer” sequences can enable researchers to specifically activate the genes that have been edited. It is also possible to add chemical tags to RNA and DNA, but it is not clear how chemical modifications to the Guide RNA and donor DNA could affect CRISPR. Here Lee et al. investigated whether adding chemical tags to the Guide RNA and/or donor DNA could enhance gene editing. The experiments show that the modified Guide RNAs and donor DNAs were still active and could edit DNA in mouse and human cells. Adding a fluorescent molecule to the donor DNA allowed Lee et al. to track which cells contained donor DNA and separate them from other cells. The fluorescent cells had twice as much editing compared to groups of unsorted cells. In further experiments, the Guide RNA and donor DNA were fused together and supplied to cells together with a DNA cutting enzyme. Cells containing this combined molecule had three times more editing than cells exposed to the original CRISPR system. This change may aid the development of new uses for CRISPR because it simplifies the system from three components (an enzyme, Guide RNA and donor DNA) to just a cutting enzyme and the combined molecule. Overall, the findings of Lee et al. show that chemical modifications to Guide RNA and donor DNA can make the CRISPR system more versatile. It opens up the possibility of new applications such as adding a targeting group that would direct the CRISPR Cas9 system to a specific cell type or tissue.
Yinong Yang - One of the best experts on this subject based on the ideXlab platform.
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genome wide prediction of highly specific Guide RNA spacers for crispr cas9 mediated genome editing in model plants and major crops
Molecular Plant, 2014Co-Authors: Jianwei Zhang, Yinong YangAbstract:Dear Editor, RNA-Guided genome editing (RGE) using the Streptococcus pyogenes CRISPR-Cas9 system (Jinek et al.,2012;Cong et al.,2013;Mali et al.,2013b) is emerging as a simple and highly efficient tool for genome editing in many organisms.The Cas9 nuclease can be programmed by dual or single Guide RNA (gRNA) to cut target DNA at specific sites,thereby introducing precise mutations by error-prone non-homologousend-joining repairing or by incorporating foreign DNAs via homologous recombination between target site and donor DNA.The gRNA-Cas9 complex recognizes targets based on the complementarity between one strand of targeted DNA (referred as protospacer) and the 5'-end leading sequence of gRNA (referred to as gRNA spacer) that is approximately 20 base pairs (bp) long (Figure 1A).Besides gRNA-DNA pairing,a protospacer-adjacent motif (PAM) following the paired region in the DNA is also required for Cas9 cleavage.Recent studies reveal that Cas9 could cut the PAM-containing DNA sites that imperfectly match gRNA spacer sequences,resulting in genome editing at undesired positions.
Daan C Swarts - One of the best experts on this subject based on the ideXlab platform.
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Preparation and electroporation of Cas12a/Cpf1-Guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells.
Methods in Enzymology, 2019Co-Authors: Lucas Kissling, Daan C Swarts, Asun Monfort, Anton Wutz, Martin JinekAbstract:Abstract CRISPR–Cas12a is a bacterial RNA-Guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-Guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of Guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. In addition, we present data illustrating the use of pairs of Cas12a nucleases for engineering large genetic deletions and outline experimental considerations for applications of Cas12a nucleases in ES cells.
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Structural basis for Guide RNA processing and seed-dependent DNA targeting and cleavage by CRISPR-Cas12a
Molecular Cell, 2017Co-Authors: Daan C Swarts, Martin Jinek, John Van Der OostAbstract: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 in a binary complex with a Guide RNA, and in a R-loop complex containing a non-cleavable Guide RNA precursor and full-length target DNA. Corroborated by biochemical experiments, these structures elucidate the mechanisms of Guide RNA processing and pre-ordering of the seed sequence in the Guide RNA that primes Cas12a for target DNA binding. The R-loop complex structure furthermore 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 that may contribute to further development of genome editing technologies.
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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.
