Real-time observation of flexible domain movements in Cas9

Osuka, Saki; Isomura, Kazushi; Kajimoto, Shohei; Komori, Tomotaka; Nishimasu, Hiroshi; Shima, Tomohiro; Nureki, Osamu; Uemura, Shunsuke

doi:10.1101/122069

Cited by 8 publications

(9 citation statements)

References 31 publications

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“…While we were submitting our manuscript, two similar studies were deposited onto bioRxiv (Dagdas et al, 2017;Osuka et al, 2017). Using smFRET approach to capture dynamic motions of the HNH domain, both studies revealed that Cas9 exhibits three major conformational states, which is consistent with the open, intermediate, and closed Cas9 states we discovered.…”

Section: Dynamic Actions Of Cas9supporting

confidence: 58%

Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

Yang

Peng

Sun

et al. 2017

Preprint

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Off-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on-and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

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Section: Dynamic Actions Of Cas9supporting

confidence: 58%

Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

Yang

Peng

Sun

et al. 2017

Preprint

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“…Specifically, smFRET has shown reciprocal changes in the conformational states assumed by HNH and REC2 across multiple DNA substrates, indicating that the conformational dynamics of HNH and REC2 is tightly coupled to ensure catalysis. Notably, the HNH domain has high conformational plasticity, as shown by biochemical and biophysical experiments (Osuka et al ., 2018; Sternberg et al ., 2015) and previous shorter MD simulations (Palermo et al ., 2016). Additionally, in the cryo-EM structure EMD-3277, the HNH and REC2 are observed at a lower resolution (8–10 Å) than the overall structure (6 Å), highlighting their mobility (Jiang et al ., 2016).…”

Section: Resultsmentioning

confidence: 99%

“…used bulk FRET to reveal that the conformational dynamics of the HNH domain controls DNA cleavage (Sternberg et al ., 2015). Subsequently, single-molecule FRET (smFRET) experiments characterized the conformational features of the activated HNH docked at the cleavage site (Dagdas et al ., 2017), and also revealed that the high flexibility of the REC lobe facilitates the activation of the HNH domain (Chen et al ., 2017; Osuka et al ., 2018). Moreover, computational studies have contributed in understanding the conformational dynamics of HNH from the apo form of Cas9 up to the DNA-bound state (Huai et al ., 2017; Palermo et al ., 2017 a , 2017 b ; Zuo and Liu, 2017).…”

Section: Introductionmentioning

confidence: 99%

Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain

Palermo

Chen

Ricci

et al. 2018

Quart. Rev. Biophys.

157

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Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)–Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 – a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution – reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1–3 regions ‘sense’ nucleic acids, ‘regulate’ the HNH conformational transition, and ultimately ‘lock’ the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1–3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

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“…However, low brightness of the luminescent probes has limited the application of luminescent imaging. Recently, several bright and multicolor Osuka et al 2018) smFRET measurements have revealed that HNH nuclease domain of Cas9 assumes two different phases: (a) a static phase in which the HNH domain remains stationary for an extended period (over 100 s) and (b) a fluctuating phase in which the domain moves frequently between multiple positions (Osuka et al 2018). Only during the fluctuating phase, the Cas9 takes structure capable to cleave DNA strands.…”

Section: Advances In Smfret Methodsmentioning

confidence: 99%

“…Thus, the ternary complex has been predicted to take additional temporal conformations beyond the solved crystal structures during DNA cleavage. Single-molecule measurements of intramolecular FRET between probes in the HNH domain and the domain proximal to the DNA cleavage site demonstrated that the ternary complex shows both static and fluctuating phases (Osuka et al 2018). In the static phase, the HNH domain stays at the DNA-undocked position, which is 3-nm away from the DNA cleavage site, for an extended period.…”

Section: Molecular Dynamics Of Proteins Measured By Smfretmentioning

confidence: 99%

Molecular Dynamics Revealed by Single-Molecule FRET Measurement

Shima

Uemura

2019

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FRET techniques have been widely used for measuring the dynamics of biomolecules because of its high sensitivity as a nanoscale distance sensor. Between two closely located fluorescent molecules, energy in an excited donor fluorescent probe is resonantly transferred to an adjacent acceptor fluorescent probe, thereby decreasing the donor's fluorescence intensity and increasing the acceptor's fluorescence intensity. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between the two fluorescent molecules. Accordingly, FRET is an extremely sensitive measurement system for detecting changes in the distance between two fluorescent probes, particularly around what is called the Förster distance, namely, a distance that yields a FRET efficiency of 0.5 (4-7 nm for a pair of typical fluorescent probes) (Lakowicz 2006). As such, FRET measurement is ideally suited for detecting changes in the distance between domains or subunits within a protein or nucleic acids during conformational changes. Moreover, based on the ratio of fluorescence intensities of two fluorescent molecules, it can achieve high signal-to-noise ratio in measurements of binding and dissociation reactions compared with measurements involving a single fluorescent molecule. These advantages have made FRET an extensively used technique for researching the dynamics of biomolecules. Conventional bulk FRET measurements, however, only yield mean measurement values of a large number of molecules. Therefore, these measurements are unable to extract information on the distribution of multiple molecules. The development of single-molecule imaging technologies, capable of distinguishing fluorescence intensities from individual molecules, has overcome this limitation. In combination with the single-molecule imaging techniques, FRET measurements are able to distinguish the state of each molecule in real time. This combination has

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Real-time observation of flexible domain movements in Cas9

Abstract: 20The CRISPR-associated protein Cas9 is a widely used genome editing tool that

Cited by 8 publications

References 31 publications

Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

Key role of the REC lobe during CRISPR–Cas9 activation by ‘sensing’, ‘regulating’, and ‘locking’ the catalytic HNH domain

Molecular Dynamics Revealed by Single-Molecule FRET Measurement

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