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cas, dna, cleavage, target, crrna, article, fret, fig, conformational, binding, google, scholar, tracrrna, casgrna, nature, rloop, conformation, complex, grnas, inactive, supplementary, singlemolecule, data, activity, min, time, nuclease, structure, state, casgrnadna, efficiency, states, crisprcas, sequences, end, results, pamdistal, ads, structural, kim, genome, open, guide, endonuclease, active, apocas, rna, fluorescence, formation, strand,

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nature portfolio privacy policy rna-related solutions national research foundation rna-programmed genome editing advertising additional information short-lived open conformation γ=δ iacceptor/δ idonor=1 social media efficient genome editing research 0/ reprints nature 471 nature 507 nature 513 nature 527 nature ni-nta agarose beads single-molecule fluorescence spectroscopy seong keun kim rna-guided endonuclease utilizing genome editing water-immersion objective lens temperature-dependent circular dichroism open conformation resulted increased-fidelity spcas9 variants rna-directed adaptive immunity crrna–dna r-loop structure author trans-encoded small rna single rna-guided cas9 single-molecule fret data supplementary references synchronous single-molecule measurement watson–crick base pairing cas9 rna-guided endonuclease peltier-type temperature controller initiate large-scale conformational r-loop structure based entire rna–dna heteroduplex dna double-helix structures single-molecule time traces prevent urea-mediated denaturation cas9–grna–dna ternary complex references jansen rna–dna base pairing single-molecule fret analysis single-molecule fret measurements pam-proximal bound complex

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         description: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. In bacteria, CRISPR-Cas9 identifies and cleaves the target DNA with the assistance of a tracrRNA and a crRNA. Here the authors use single-molecule spectroscopy to investigate conformational changes and show that the tracrRNA keeps Cas9 in a functionally active form.
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      headline:Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
      description: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. In bacteria, CRISPR-Cas9 identifies and cleaves the target DNA with the assistance of a tracrRNA and a crRNA. Here the authors use single-molecule spectroscopy to investigate conformational changes and show that the tracrRNA keeps Cas9 in a functionally active form.
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