Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

doi:10.1038/s41467-017-01466-8

Cited by 200 publications

(213 citation statements)

References 31 publications

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“…In the studies by Dagdas et al and Chen et al , most of the S355C–S867C molecules exhibited ~1.0 FRET efficiency, suggesting that the HNH domain in almost all of the Cas9 molecules adopts the cleavage‐competent (D) position. In contrast, in the studies by us and Yang et al , the major peak value of the FRET efficiency was ~0.4, suggesting that the HNH domain is predominantly located in the intermediate (I) position, consistent with the recent HS‐AFM observation (Shibata et al , ). In addition, the studies by us and Yang et al demonstrated the existence of the pre‐cleavage (D*) HNH position.…”

Section: Discussionsupporting

confidence: 83%

“…Our smFRET data revealed the fluctuations between the REC and NUC lobes in apo‐Cas9 (Fig ), indicating that, in addition to the static closed conformation, apo‐Cas9 adopts transient conformations. Consistent with this, a single‐molecule study using high‐speed atomic force microscopy (HS‐AFM) visualized highly flexible conformations of apo‐Cas9 (Shibata et al , ). The movement of the REC lobe relative to the NUC lobe can provide interaction interfaces for the sgRNA; thus, the flexible movement in apo‐Cas9 may play an important role in the assembly with the sgRNA.…”

Section: Discussionmentioning

confidence: 79%

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Real‐time observation of flexible domain movements in CRISPR–Cas9

et al. 2018

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The CRISPR‐associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single‐guide RNA (sgRNA). Structural studies have revealed the multi‐domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA. These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single‐molecule FRET. We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo‐Cas9, which may play a role in the assembly with the sgRNA. Collectively, our results highlight the potential role of domain fluctuations in driving Cas9‐catalyzed DNA cleavage.

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 79%

Real‐time observation of flexible domain movements in CRISPR–Cas9

et al. 2018

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“…5F). It has been shown that Cas9 can remain bound to the DNA after cleavage for an extended time [33][34][35] and it preferentially releases the PAM-distal, non-target strand 36 . Our data suggests that this property might result in an asymmetrical influence of the bound Cas9/gRNA complex on the HDR process, and that perhaps the released PAM-distal, non-target strand may promote efficient HDR.…”

Section: Impaired Homology Asymmetrically Affects Drive Efficiencymentioning

confidence: 99%

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

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Bishop

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et al. 2019

Preprint

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CRISPR-based gene drives spread through populations bypassing the dictates of Mendelian genetics, offering a population-engineering tool for tackling vector-borne diseases, managing crop pests, and helping island conservation efforts; unfortunately, current technologies raise safety concerns for unintended gene propagation. Herein, we address this by splitting the two drive components, Cas9 and gRNAs, into separate alleles to form a novel trans-complementing split-genedrive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This bi-component nature allows for individual transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a smallmolecule-controlled version to investigate the biology of component inheritance and use our system to study the maternal effects on CRISPR inheritance, impaired homology on efficiency, and resistant allele formation. Lastly, mathematical modeling of tGD spread in a population shows potential advantages for improving current gene-drive technologies for field population modification. Figure 1 -The trans-complementing gene-drive system (tGD) features simultaneous super-Mendelian inheritance of two transgenes.(A) Schematic of the tGD genetic arrangement with two elements that can be kept separated as different transgenic lines. The Cas9 transgene is inserted in the genomic location targeted by the gRNA-A, while a separate cassette expressing a tandem gRNA construct (gRNA-A, gRNA-B) is inserted at the location targeted by gRNA-B. Upon genetic cross, each gRNA combines with Cas9 to generate a double-strand DNA break at each locus on the wild-type allele. Each break is then repaired by homology-directed repair (HDR) pathway using the intact chromosome carrying the transgene as a template. (B) Outline of the genetic cross used to demonstrate tGD in fruit flies. F0 males carrying a DsRed-marked Cas9 transgene inserted in the yellow locus were crossed to females carrying a GFP-marked cassette containing two gRNAs (y1-e1) inserted in the ebony coding sequence. Trans-heterozygous F1 females (carrying both Cas9 and gRNAs) were crossed to wild-type males to assess germline transmission rates of the fluorophores marking the transgenes in the F2 progeny. The conversion event is indicated by the red and green triangles in the F1 females (C) Single F1 female germline inheritance output measured as GFP and DsRed marker presence in the F2 progeny. The black bar represents the inheritance average. The blue shading represents the deviation from the expected 50% "Mendelian" inheritance. Inheritance average, standard deviation, number of samples (n) and total number of flies scored in each experiment are represented over the graph in line with the respective data.

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“…The Cas9-RNA complex interrogates the target sites on the DNA via 3D diffusion, and recognises the complementary target site with the NGG PAM diffusion (Shibata et al, 2017). We used time-lapse microscopy to visualise the real-time dynamics of the dCas9-RNA complex for the labelling of telomeres of fixed nuclei in action (Fig.…”

Section: Researchmentioning

confidence: 99%

RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

et al. 2019

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Summary Visualising the spatio‐temporal organisation of the genome will improve our understanding of how chromatin structure and function are intertwined. We developed a tool to visualise defined genomic sequences in fixed nuclei and chromosomes based on a two‐part guide RNA with a recombinant Cas9 endonuclease complex. This method does not require any special construct or transformation method. In contrast to classical fluorescence in situ hybridiaztion, RGEN ‐ ISL ( RNA ‐guided endonuclease – in situ labelling) does not require DNA denaturation, and therefore permits a better structural chromatin preservation. The application of differentially labelled trans ‐activating cr RNA s allows the multiplexing of RGEN ‐ ISL . Moreover, this technique is combinable with immunohistochemistry. Real‐time visualisation of the CRISPR /Cas9‐mediated DNA labelling process revealed the kinetics of the reaction. The broad range of adaptability of RGEN ‐ ISL to different temperatures and combinations of methods has the potential to advance the field of chromosome biology.

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Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

Cited by 200 publications

References 31 publications

Real‐time observation of flexible domain movements in CRISPR–Cas9

Real‐time observation of flexible domain movements in CRISPR–Cas9

Split-gene drive system provides flexible application for safe laboratory investigation and potential field deployment

RNA‐guided endonuclease – in situ labelling (RGEN‐ISL): a fast CRISPR/Cas9‐based method to label genomic sequences in various species

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