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072edcdc5d9e6d23b191954e7276feb43536b7f7793a6e3f5edbaef397953ad2/preprint/images_list.json

@@ -0,0 +1,42 @@
+[
+    {
+        "type": "image",
+        "img_path": "images/Figure_1.png",
+        "caption": "Native-PAGE analysis to elucidate Cas9 targets in the nucleosome.\n(a) Schematic of the experimental setup for in vitro binding and cleavage assays. The sgRNA that recognizes each target site was incubated with Cy5-labeled SpCas9-NG mutant that recognizes the NG PAM sequence. Mononucleosomes contained Cy3-labeled histone H4 and 5' -end-labeled DNA with 6 FAM were then added to Cas9-sgRNA ribonucleoproteins. The 5' -end-labeled DNA is colored light purple, and the non-labeled DNA is colored dark purple. Histones H2A, H2B, H3, and H4 are colored yellow, light red, light blue, and light green, respectively.\n(b) Schematic of the nucleosome DNA containing the Widom 601 sequence. The location of the histone core is indicated by the light gray oval. The location of PAMs within the double-stranded sequence are indicated with arrows, pointing in the 5' to 3' orientation of the NG sequence. The dark red and the dark blue arrows show the NG sequences on the solvent and histone sides, respectively. \u00a06 FAM is attached to the 5' end of the left side of the DNA.\n(c and d) \u00a0In vitro nucleosome DNA cleavage activities of the SpCas9-NG for each PAM sequence. The reaction products were resolved, visualized with an Amersham Imager 680 (Cytiva). Data are mean \u00b1 s.d. (n = 2). The experiments were repeated three times with similar results. Source data is provided as a Source data file.\n(e and f) Native-PAGE gels showing the results of a binding assay targeting the indicated PAMs. The native-PAGE gels were imaged in merged three-color channels: 6-FAM on the nucleosome DNA end (blue), Cy3 on histone H4 (green) and Cy5 on Cas9 (red). The results of PAM1 to PAM15 and PAM16 to PAM28 are shown in (e) and (f), respectively.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_2.png",
+        "caption": "Cryo-EM structure of Cas9-sgRNA-nucleosome complex targeting linker DNA.\n(a) The domain organization of S. pyogenes Cas9. Residues 178 to 297 and 766 to 924 were not included in the final model.\n(b) Cryo-EM density maps of the Cas9-sgRNA-nucleosome complex in DNA attached state.\n(c) Overall structure of the Cas9-sgRNA-nucleosome complex in DNA attached state. Disordered regions are indicated as dotted lines.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_3.png",
+        "caption": "Suggested interactions between Cas9 and nucleosome.\n(a) \u00a0Close-up view of the suggested Cas9-histone tail interacting regions. The cryo-EM density enclosed by a dashed oval indicates the EM density of the histone tail.\n(b) In vitrobinding activities of the wild-type Cas9 for canonical nucleosomes and tail-less nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37\u2009\u00b0C for 10 min. The Cy5 fluorescence of Cas9 in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean \u00b1 s.d. (n = 3). The experiments were repeated three times with similar results.\n(c) In vitro nucleosome DNA cleavage activities of the wild-type Cas9 for canonical nucleosomes and tail-less nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37\u2009\u00b0C for 10 min. The EtBr stained reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean \u00b1 s.d. (n = 3). The experiments were repeated three times with similar results.\n(d) \u00a0Close-up view of the suggested interacting regions of the PI edge and nucleosomal DNA. The lysine (K) residues that potentially contact the DNA are highlighted.\n(e) In vitrobinding activities of the wild-type Cas9 and Cas9 mutants to reduce interaction between PI edge and nucleosome DNA entry. The Cy3 fluorescence of nucleosomes in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean \u00b1 s.d. (n = 3). The experiments were repeated three times with similar results.\n(f) In vitro nucleosome DNA cleavage activities of the wild-type Cas9 and Cas9 mutants to reduce interaction between PI edge and nucleosome DNA entry. The 6-FAM fluorescence of nucleosomes in reaction products were visualized by Native-PAGE analysis with an Amersham Imager 680 (Cytiva), and quantified by ImageJ. Data are mean \u00b1 s.d. (n = 3). The experiments were repeated three times with similar results.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_4.png",
+        "caption": "Cas9-core DNA interaction affect nucleosome DNA cleavage efficiency in vitro\n(a) Close-up view of the suggested interactions between the PI domain and nucleosomal core DNA. The lysine (K), arginine (R) and histidine (H) residues that potentially contact the DNA are highlighted.\n(b) In vitro binding activities of wild-type Cas9 and Cas9 mutants reducing interaction between Cas9 and core DNA for nucleosomes. The nucleosome was incubated with the Cas9-sgRNA complex at 37\u2009\u00b0C for 10 min. The Cy3 fluorescence of nucleosomes in the reaction product was resolved and visualized by Native-PAGE analysis, and quantified by ImageJ. Data are mean \u00b1 s.d. (n =\u20093 biologically independent samples). The experiments were repeated three times with similar results.\n(c) In vitro nucleosome DNA cleavage activities of wild-type Cas9 and mutants reducing interaction between Cas9 and core DNA. The nucleosome was incubated with the Cas9-sgRNA complex at 37\u2009\u00b0C for 30 min. The 6-FAM fluorescence of nucleosomes in the reaction product was resolved and visualized by Native-PAGE analysis, and quantified by ImageJ. Data are mean \u00b1 s.d. (n =\u20093 biologically independent samples). The experiments were repeated three times with similar results.\n(d) Schematic of the experimental setup for in vitro binding and cleavage assays.\n(e) In vivo mutation efficiencies at three target sites (SPL7, SPL17 and GAPDH) in transgenic rice calli of the wild-type Cas9 and Cas9 mutants reducing interaction between Cas9 and core DNA for nucleosomes. Dots indicate the mutation frequency for each independent calli. Bars indicate the average mutation frequency. Error bars indicate the standard error (SE).",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_5.png",
+        "caption": "Mechanism of nucleosome-mediated prevention of DNA cleavage by Cas9.\nIn the first step, Cas9 is restricted from accessing the target sequence by the flexibility of the DNA end regions. Subsequently, even if Cas9 overcomes the restriction and binds to the target sequence, non-specific interactions with the core DNA reduces helicase activity of Cas9 leading to the decrease of DNA cleavage efficiency.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    }
+]

072edcdc5d9e6d23b191954e7276feb43536b7f7793a6e3f5edbaef397953ad2/preprint/preprint.md

@@ -0,0 +1,185 @@
+# Abstract
+
+The CRISPR-associated endonuclease Cas9 is used as a genome editing tool, which targets specific genomic loci by single guide RNAs (sgRNAs). In eukaryotes, genome DNA is stored in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we found that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but does not target the DNA tightly wrapped around the histone octamer. We determined cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggested interactions between Cas9 and nucleosomes at multiple sites, and mutants that reduced the interaction between nucleosomal DNA and Cas9 improved nucleosomal DNA cleavage activity both in vitro and in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.
+
+[Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy](/browse?subjectArea=Biological%20sciences%2FStructural%20biology%2FElectron%20microscopy%2FCryoelectron%20microscopy)
+
+[Biological sciences/Molecular biology/CRISPR-Cas systems/CRISPR-Cas9 genome editing](/browse?subjectArea=Biological%20sciences%2FMolecular%20biology%2FCRISPR-Cas%20systems%2FCRISPR-Cas9%20genome%20editing)
+
+# Main
+
+CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated) systems in bacteria provide adaptive immunity against foreign nucleic acids<sup>1</sup>. Cas9 associates with either dual RNA guides (CRISPR RNA [crRNA] and a trans-activating crRNA [tracrRNA]) or a combined single guide RNA (sgRNA). These RNA modules direct Cas9 to target specific DNA sequences, facilitating the cleavage of double-stranded DNA. The target DNA is complementary to an approximately 20 nt guide segment in the guide RNA and is flanked by a protospacer adjacent motif (PAM). Cas9 from *Streptococcus pyogenes* especially exhibits robust nuclease activities in eukaryotic cells, and is widely used as versatile genome-engineering tool<sup>2</sup>.
+
+CRISPR-Cas9 systems are derived from bacteria with genomes typically composed of supercoiled circular DNA with nucleoid-associated proteins<sup>3</sup>. In contrast, eukaryotes, the targets of genome editing, store genome DNA in chromatin, in which the primary structure is the nucleosome<sup>4,5</sup>. The nucleosome contains four histone proteins, H2A, H2B, H3, and H4, which form a histone octamer that is wrapped by 145~147 bp DNA<sup>6</sup>. In the nucleosome, the DNA directly interacting with histones (core DNA) extends linker DNAs, which are histone-free DNA regions next to both sides of the nucleosomal DNA ends.
+
+Various structural analyses have sought to clarify the DNA cleavage mechanism of Cas9, but all have focused on how Cas9 cleaves free DNA<sup>7–12</sup>. However, due to a lack of structural insights into Cas9 targeting DNA in nucleosomes, the principles governing Cas9 targeting the eukaryotic genome remain unclear. In addition, nucleosomes reportedly suppress Cas9 DNA cleavage both *in vitro* and *in vivo*<sup>13–15</sup>, indicating that many genomic DNA sites escape Cas9 targeting. This problem has largely limited the applications of Cas9 for eukaryotic genome editing, which has not been resolved due to the lack of structural information about Cas9 and nucleosome interactions.
+
+## Cas9 targets nucleosomal DNA end regions
+
+To elucidate where Cas9 binds and cleaves the DNA in the nucleosome, we performed a native-polyacrylamide gel electrophoresis (PAGE) analysis (Fig.1a).
+
+The reconstituted nucleosome contained Cy3-labeled histone H4 and 6 FAM end-labeled DNA consisting of a Widom 601 positioning sequence<sup>16</sup> for core nucleosomal DNA (histone-DNA contacting region) with 25 base pairs of linker DNA (histone-free DNA region) on both sides (Fig.1a, b). The wild-type (WT) Cas9 recognizes NGG sequences to cleave DNA, limiting the number of targetable DNA regions on the nucleosome DNA sequence. To overcome this limitation, we utilized the SpCas9-NG that recognizes the NG PAM sequence<sup>17</sup>, enabling the targeting of 28 sites (PAM1–PAM28) in each superhelical locations (SHLs) in the nucleosome core particle and linker DNA regions (Fig. 1b and Extended data Fig. 1a).
+
+The native-PAGE analysis showed that Cas9 cleaved the linker DNA (at PAM1 and PAM28) (Fig. 1c, d), consistent with the previous studies<sup>18–20</sup>. In addition, DNA cleavage by Cas9 at SHL+6 (PAM27) was observed in the core DNA region (Fig. 1d). By contrast, DNA binding and cleavage by Cas9 were not observed at SHL0–SHL±5 (PAM2–PAM14 and PAM19–PAM26) (Fig. 1c, d, e, f). A previous study reported that the DNA region around SHL±6 to SHL±7 transiently unwrapped from the histone surface (DNA unwrapping)<sup>21</sup>, while the histone-DNA contact is stable at SHL0–SHL±5<sup>22</sup>. Therefore, DNA unwrapping may enable Cas9 to bind and cleave the region from SHL+6 to the linker DNA, whereas stronger histone and DNA interactions may prevent Cas9 from even binding at SHL0–SHL±5.
+
+Despite the nucleosome’s symmetrical structure with SHL0 as the central axis, the native-PAGE analysis also showed that the efficiency of nucleosome DNA cleavage by Cas9 significantly differs between the DNA entry/exit sites (Fig. 1c, d). Specifically, the DNA cleavage activity was lower for PAM1 (at DNA entry side) compared to PAM28 (at DNA exit side), and the DNA cleavage observed at SHL+6 (PAM27) was not observed at SHL-6 (PAM18) (Fig. 1c, d and Extended data Fig. 1a). Previous studies reported that the Widom601 sequence has more flexibility on the DNA exit side across the dyad than on the DNA entry side, depending on the internal base sequence of the core DNA<sup>29–31</sup>. Thus, flexibility differences at the DNA entry/exit sites, probably caused by the base sequences of the core DNA, may impact Cas9’s DNA cleavage activity.
+
+There was no cleavage at PAM14 and PAM17 although they are in SHL7, where nucleosome unwrapping occurs. Based on the superimposed model of Cas9 and nucleosome (Extended data Fig. 1b, c), the position of Cas9 may make the capability of nucleosome DNA cleavage. In Extended data Fig. 1b, the Cas9 crushes to the histone core, making it difficult to access PAM14 and PAM17. Additionally, SHL5 is the region where the interaction between histone and DNA is strong, causing Cas9 not to pull away for DNA cleavage.
+
+## Cryo-EM analysis for Cas9-sgRNA-nucleosome complex targeting linker DNA
+
+Unexpectedly, we observed ternary complex formation of Cas9, sgRNA, and nucleosome in PAM1 (Fig. 1e). The 6-FAM fluorescence on the 5' end of the DNA was absent, indicating that the ternary complex probably represented a post-DNA cleavage state (Extended data Fig. 3). In PAM28, the formation of the ternary complex formation was not observed, which was presumably due to Cas9 departing from the nucleosome after cleaving the nucleosome DNA (Extended data Fig. 3). Therefore, the formation of the ternary complex in PAM1 is anticipated to be due to the presence of an unknown interaction between the nucleosome and Cas9.
+
+To investigate how Cas9 targets nucleosomes to form the complex, we performed a cryo-EM analysis of the Cas9-sgRNA-nucleosome complex targeting the PAM1 site. We used wild type Cas9, instead of SpCas9-NG, for this cryo-EM analysis because of the higher reconstitution efficiency of the complex. During the data processing, we observed fluctuations in the proximity of Cas9 to the nucleosome (Extended data Fig. 4 and Extended Table 1). We determined the structure of the Cas9-sgRNA-nucleosome complex in a state where Cas9 contacts the core DNA, which we refer to as the "DNA attached state" (Fig. 2).
+
+In the present structure, the HNH and REC2 domains were mostly disordered, resembling the post-DNA cleavage state (Fig. 2a, c and Extended data Fig. 5a, b), as previously reported<sup>10,23</sup>. Furthermore, no densities of the non-target DNA strand and the phosphate backbone in the target DNA strand at the cleavage site near the bridge helix were observed, indicating the double strand cleavage of nucleosomal DNA by Cas9 (Extended data Fig. 5c, d). Therefore, these findings indicated that the complex was in a post-DNA cleavage state, consistent with the results of biochemical experiments (Extended data Fig. 3).
+
+Within the complex, approximately 15 base pairs of the nucleosome DNA were peeled off from the histone core exposing the N-terminal α-helix in H3 to the solvent (Extended data Fig. 6a, b). The length of the detached DNA corresponded to the length of DNA that dissociates from the histone core during nucleosome DNA unwrapping. Therefore, Cas9 may bind to the nucleosome upon DNA unwrapping, and maintain the DNA detachment from histones, preventing the DNA from rewrapping around histones. Alternatively, Cas9 binding may induce the nucleosome DNA unwrapping around the entry-exit site. This binding mode, which maintains the DNA dissociation histones, bears similarity to the binding mode observed in previously reported complex structures of endogenous nucleosome-binding proteins, such as RNA polymerase, and nucleosomes<sup>24–26</sup> (Extended data Fig. 6c).
+
+To our knowledge, there have been no previous reports of bacterial proteins binding to nucleosomes in such a manner that partially alters the nucleosome structure. This observation leads us to speculate that this binding mode may be one of the reasons why Cas9 is functional in eukaryotic cells.
+
+## Interactions between PI domain in Cas9 and nucleosome
+
+The Cas9-sgRNA-nucleosome structure suggested several interactions between the PI domain in Cas9 and nucleosome (Fig. 3).
+
+A weak EM density was observed in the space between Cas9 and the nucleosome, which appears to be an H2A histone-tail (Fig. 3a). Although, we could not identify which residues of the histone tail interact with Cas9 due to the poor density, the histone tail is positively charged, while the Cas9 PI region likely interacting with the histone tail exposes acidic residues (Extended data Fig. 8a), suggesting that the histone tail and Cas9 electrostatically interact. To examine the effects of the histone tail on the binding and DNA cleavage efficiency of Cas9, we performed native-PAGE analyses using tail-less nucleosomes, in which the histone tails were removed. Unexpectedly, there were no significant changes in the binding affinity or DNA cleavage activity of WT Cas9 against tail-less nucleosomes compared to canonical nucleosomes (Fig. 3b, c). These results suggested that the interaction between the histone tail and the PI domain occurs after complex formation and does not play a role in Cas9's recognition of the target sequence within the nucleosome.
+
+The loop between the two β-sheets (β18-β19) of the PI domain, referred to as the PI edge in this study, were suggested to interact with the DNA near the DNA entry site (Fig. 3d). The PI edge contains numerous lysine residues, which were implemented to recognize the phosphate backbone of DNA (Fig. 3d). To investigate the impact of the PI edge on the stability of the complex and Cas9’s DNA cleavage activity, mutations were introduced at K1151, K1153, K1155, and K1156. Native-PAGE results showed a reduction in complex formation for the mutants including K1155E (Fig. 3e), suggesting that K1155 is crucial for the stability of the Cas9-sgRNA-nucleosome complex obtained in this study. In contrast, there were no significant differences in DNA cleavage efficiencies between the WT and mutants (Fig. 3f). After DNA cleavage, Cas9 reportedly moves away from the DNA, including the PAM<sup>27</sup>. Therefore, while Cas9 would generally dissociate after cleavage, Cas9 entanglement in the nucleosome through the PI edge may lead to continuous complex formation after DNA cleavage.
+
+## Nonspecific interactions between Cas9 and core DNA affects DNA cleavage efficiency *in vitro* and *in vivo*
+
+Two loops, bridging helices 49 and 50 and helices 51 and 52 in the PI domain, were suggested to associate with the phosphate backbone of the core DNA (Fig. 4a). Mutations were introduced into five basic residues (H1264, K1296, H1297, R1298, and K1300) in these loops. We then explored their effects on the binding affinity and DNA cleavage activity towards the nucleosome at PAM1 using native-PAGE analyses (Fig. 4b, c). Interestingly, mutants incorporating H1264, R1298, and K1300 exhibited increases in nucleosome binding and DNA cleavage activities (Fig. 4b, c). Given that Cas9-sgRNA-nucleosome complex formed in PAM1 was in post-DNA cleaved state, it is plausible that binding and cleavage activities increase simultaneously. These results suggested that the cleavage of nucleosomal DNA may be hindered by non-specific interactions between Cas9 and core DNA *in vitro*, even in the regions where Cas9 can cleave DNA.
+
+In the proximal CRISPR (proxy-CRISPR) method, the binding of catalytically inactive Cas9 to proximal regions reportedly induces changes in the chromatin structure of the target locus, thus enhancing the genome editing efficiency of another nuclease-active Cas9<sup>28</sup>. Therefore, we conducted mutagenesis in the rice callus by targeting regions where the proxy-CRISPR method enhanced mutation efficacy, as they are presumed to bear chromatin and nucleosome structures (Fig. 4d). The Cas9 mutants that reduced interaction between the PI domain and core DNA induced mutations at efficiencies similar to or higher than those of WT Cas9 (Fig. 4e). It was surprising that the mutants designed to target a specific position within linker DNA exhibited activity even in vivo. Factors contributing to the limited improvement in genome editing activity *in vivo* compared to *in vitro* results may include the non-static nature of nucleosome positioning due to as the cell cycle, as well as the potential limitation of the designed mutant in a more dynamic *in vivo* environment, which contrasts with the controlled conditions *in vitro*. Therefore, although the Cas9 mutants have a constrained efficacy in the more dynamic conditions present *in vivo*, these results indicated that the interaction between the PI domain and core DNA may negatively influence genome editing efficiency *in vivo*.
+
+# Discussion
+
+In this study, our native-PAGE analysis showed that Cas9 can target nucleosome DNA end regions, linker DNA and SHL+6 regions. The native-PAGE analysis also revealed significant differences in the efficiency of Cas9-mediated cleavage of nucleosomal DNA between the DNA entry and exit sites. It is hypothesized that these differences are attributable to the varying flexibility of the nucleosomal DNA between the entry and exit sites<sup>29–31</sup>, which in turn may affect Cas9 accessibility. Thus, flexibility differences at the DNA entry/exit sites, probably caused by the base sequences of the core DNA, may impact Cas9’s DNA cleavage activity. Additionally, since the flexibility of the DNA near the ends of the nucleosomal DNA affected the cleavage efficiency of linker DNA, it is possible that even if target sites are set to avoid nucleosome positions, the sequence and flexibility of the DNA within nearby nucleosomes could control Cas9 access and thereby influence genome editing activity. Future research is expected to elucidate these factors.
+
+We further determined the structure of the Cas9-sgRNA-nucleosome complex in a post-cleaved state. The structure revealed several non-specific interactions between Cas9 and the nucleosome. The native-PAGE analysis and genome-editing analysis in rice calli by Cas9 mutants suggested that the interaction between the PI domain and core DNA may especially hinder Cas9 DNA cleavage both<em>in vitro</em> and<em>in vivo</em>.
+
+Based on these observations, we propose the following mechanism for the nucleosome-mediated inhibition of DNA cleavage by Cas9 (Fig. 5). The inhibition occurs in two stages. First, Cas9 is restricted from accessing the target sequence in nucleosomes by the low flexibility of the DNA end regions. In living organisms, the flexibility of the nucleosome DNA end, which is regulated by the internal sequence of the nucleosomal DNA and post-translational modifications (PTMs), controls the interactions of nucleosome-binding factors<sup>32,33</sup>. Thus, the DNA accessibility of Cas9 may also be regulated by the nucleosome DNA end flexibility. Second, even if Cas9 overcomes the restriction and binds to the target sequence, non-specific interactions with nucleosomal DNA reduce the motions of Cas9 needed for DNA cleavage, thus decreasing the DNA cleavage efficiency. The specific manner by which the non-specific interactions hinder Cas9 and the inhibited entity are presently unclear. However, given that the PI domain is involved in PAM sequence recognition, it is possible that the interaction of the PI domain may influence PAM recognition.
+
+Diverse types of Cas effectors, each exhibiting significant variations in their structures and properties, have been discovered<sup>34</sup>. The mechanisms of nucleosome access and DNA cleavage activities are also anticipated to diverge among these Cas effectors. The experimental series conducted in this study, ranging from the exploration of binding and DNA cleavage sites on nucleosomes to structural analysis, can be applied to other Cas effectors. This approach may clarify the interplay between various Cas effectors and nucleosomes, leading to the development of novel genome editing tools. This study offers a new perspective on the development of genome editing tools by highlighting the interaction between Cas proteins and chromatin structures.
+
+# Methods
+
+## Expression and purification of proteins
+
+The genes encoding full-length *S. pyogenes* Cas9 (residues 1–1368) and *S. pyogenes* Cas9 mutant R1335V/G1288R/T1337R/L1111R/A1322R/E1219F/D1135V (NG SpCas9) <sup>17</sup> were separately cloned into a pET28 vector with an N-terminal hexa-histidine (His6) tag. The WT SpCas9 and SpCas9 mutant were expressed at 20°C in *Escherichia coli* Rosetta 2 (DE3) (Novagen), and purified by chromatography on Ni-NTA Superflow resin (QIAGEN). The eluted protein was incubated overnight at 4°C with TEV protease to remove the His<sub>6</sub>–tag, and further purified by chromatography on Ni-NTA, Hitrap SP HP (GE Healthcare) and HiLoad Superdex 200 16/60 (GE Healthcare) columns in buffer consisting of 20 mM Tris-HCl (pH 8.0) with 150 mM NaCl.
+
+Recombinant histone proteins were prepared as described previously <sup>25</sup>. For the preparation of fluorescent labeled nucleosomes, E63 in H4 was substituted with C. Briefly, histone proteins H2A, H2B, H3, and H4 were expressed as His6-tagged proteins in Escherichia coli cells, and then purified by Ni-NTA column chromatography (Qiagen) under denaturing condition. After His6 tag cleavage by thrombin protease (Wako), the resulting histone proteins were further purified by MonoS cation exchange column chromatography (GE Healthcare). The purified histone proteins were lyophilized and stored at 4°C.
+
+## Nucleosome reconstitution
+
+Histone octamers were reconstituted with purified H2A, H2B, H3, and H4 as described previously <sup>25</sup>. The 193 bp Widom 601 DNA <sup>16</sup> was prepared as described previously <sup>35</sup>. Nucleosomes containing the 193 bp Widom 601 DNA with 6-FAM were reconstituted by the salt dialysis method, and purified by non-denaturing electrophoresis using a Prep Cell apparatus (Bio-Rad) as described previously (all other experiments) <sup>25</sup>. The purified nucleosomes were dialyzed in 20 mM Tris-Cl buffer (pH7.5) containing 1 mM DTT and 5 % glycerol. The resulting nucleosomes were flash-frozen in liquid nitrogen and stored at -80 °C.
+
+## Preparation of fluorescent proteins
+
+Cas9 was mixed with a three-fold molar excess of Cy5-NHS-ester (Funakoshi), and further incubated at 4°C for 2h. Subsequently, Cy5-labeled Cas9 was purified by HiLoad Superdex 200 16/60 (GE Healthcare) columns in buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl) (Extended data Fig. 2).
+
+E63C in H4 was labeled with Cy3-maleimide (Funakoshi). After labeling, H2A, H2B, H3, and H4 were mixed under denaturing conditions. The mixture was dialyzed against high-salt buffer, and the octamer was reconstituted and purified by Superdex 200 gel filtration chromatography.
+
+For DNA amplification, a 193 bp DNA containing the Widom 601 sequence was PCR-amplified using a primer labeled with 6-FAM at the 5' end synthesized by FASMAC. The product was then phenol-chloroform extracted, ethanol precipitated, and purified by non-denaturing PAGE.
+
+Subsequently, the histone octamer and DNA were mixed, and the complex was reconstituted by salt dialysis as described above. Nucleosomes were reconstituted and further purified by non-denaturing PAGE.
+
+## Single-guide RNA (sgRNA) preparation
+
+With respect to the location of PAM sequences in the nucleosomal DNA, we designed sgRNA sequences to target PAM sequences present on at least one histone side and one solvent side at each super helix location (SHL) in the nucleosome (Fig. 1b and Extended data Fig. 1a). The 100-nt sgRNA was transcribed in vitro with T7 polymerase using a DNA template purchased from Eurofins Genomics, and was purified by 10% denaturing polyacrylamide gel electrophoresis. The sequences of sgRNAs used in this study are listed in Supplementary Table 1.
+
+## Native-PAGE analyses
+
+Nucleosome, sgRNA, and Cas9 (WT/NG/Mutants) binding assays for the gel shift assays in this study were all conducted in the same manner. First, the complete Cas9-sgRNA ribonucleoprotein complex was formed by incubating a two-fold molar excess of the chosen sgRNA with Cas9 at room temperature for 5 min. Next, 0.5 µM DNA substrate (either naked DNA or nucleosome) was mixed with Cas9 and sgRNA (molar ratio, 1:3:6) in buffer containing 10 mM Tris-HCl pH 8.0, 15 mM NaCl, 5 mM MgCl<sub>2</sub>, and 1 mM DTT. For the detection of Cas9-mediated DNA cleavage, the Cas9-sgRNA ribonucleoprotein (RNP) incubated with nucleosomes was deproteinized by Proteinase K treatment at 37°C for 30 min. Products were separated on a native 5% polyacrylamide gel with 0.5x TBE as the running buffer. Cy3, Cy5 and 6 FAM fluorescence signals were scanned by using Amersham Typhoon scanner (Cytiva) before gels were stained with ethidium bromide.
+
+## Preparation of the Cas9-sgRNA-nucleosome complex for cryo-EM
+
+We utilized WT SpCas9 instead of the SpCas9-NG mutant for cryo-EM analysis because of the higher complex reconstruction efficiency. For the preparation of SpCas9-sgRNA-nucleosome complexes crosslinked for single particle analysis, the SpCas9-sgRNA-nucleosome binding reaction was performed by mixing nucleosome (0.5 μM), SpCas9 (2 μM), and sgRNA (2 μM) in 2 mL of reaction solution (18 mM Tris-Cl (pH 7.5), 30 mM NaCl, 1.2 mM DTT, 5 mM MgCl<sub>2</sub> and 1% glycerol) at 37 °C for 30 min. To stabilize and purify the Cas9-sgRNA-nucleosome complex, the reaction mixture was fractionated by the GraFix method <sup>36</sup>. Gradient solutions were prepared using low sucrose concentration solution (10 mM HEPES-NaOH (pH 7.5), 20 mM NaCl, 1 mM DTT, and 5% sucrose) and high sucrose concentration solution (10 mM HEPES-NaOH (pH 7.5), 20 mM NaCl, 1 mM DTT, 20 % sucrose, and 1 % glutaraldehyde [diluted from glutaraldehyde solution, Electron Microscopy Sciences, 16220]). Half of the reaction mixture was loaded on the top of the gradient solution. The sample was centrifuged at 27,000 rpm at 4 °C for 16 h using a Beckman SW41 rotor. After centrifugation, 750 μL fractions were taken from the top of the gradient, and the fractions were analyzed by Native-PAGE. The Cas9-sgRNA-nucleosome complex sample was dialyzed in buffer containing 20 mM HEPES-NaOH (pH 7.5) and 1 mM DTT. The resulting samples were concentrated using Amicon Ultra 30K filters (Merck Millipore). The DNA concentrations of the Cas9-sgRNA-nucleosome complexes plunge frozen for cryo-EM was 367.7 μg/mL.
+
+## Electron microscopy and data collection
+
+The purified complex solution was applied to freshly glow-discharged Cu/Rh 200 mesh R1.2/1.3 grids (Quantifoil), in a Vitrobot Mark IV (FEI) at 16 °C with a blotting time of 4 s under 100% humidity conditions.
+
+The cryo-EM data were collected using a Titan Krios G3i microscope (Thermo Fisher Scientific), running at 300 kV and equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector in the electron counting mode. Each movie was recorded at a nominal magnification of 81,000 x, corresponding to a calibrated pixel size of 1.05 Å (The University of Tokyo, Japan) at the electron exposure of 11.59 e/pix/sec for 5.656 s, resulting in an accumulated exposure of 54.5 e/ Å<sup>2</sup>. The data were automatically acquired by the image shift method using the SerialEM software <sup>37</sup>, with a defocus range of 1.0 to 2.5 mm, and 7,875 movies were acquired. The dose-fractionated movies were subjected to beam-induced motion correction and dose-weighting, using the MotionCor2 <sup>38</sup> algorithm implemented in RELION-3.1 <sup>39</sup>, and the contrast transfer function (CTF) parameters were estimated using CTFFIND4 <sup>40</sup>.
+
+## Electron microscopy data processing
+
+All cryo-EM data processing steps were conducted using the cryoSPARC v3.2.0. software package <sup>41</sup> (fig. S5). From the motion-corrected and dose-weighted micrographs, 4,459,962 particles were automatically picked using Blob Picker in cryoSPARC. The particles were subjected to several rounds of reference-free 2D classifications, followed by cryoSPARC Ab-Initio Reconstruction to curate particle sets. The 1,087,299 particles were further curated by cryoSPARC Heterogenous Refinement (N = 3), using the map derived from the cryoSPARC Ab-Initio Reconstruction as the template. The selected 305,595 particles were further subjected to 3D variability analysis <sup>42</sup>. The resulting maps with different conformations were refined using non-uniform refinement <sup>43</sup>, yielding maps at resolutions of 4.56 Å, according to the FSC criterion of 0.143 <sup>44</sup>. The local resolution was estimated with BlocRes in cryoSPARC.
+
+## Model building
+
+Rigid docking was performed in COOT <sup>45</sup> using the crystal structures of the Cas9-sgRNA-target DNA complex (PDB code 4UN3) and the nucleosome (PDB code 3LZ0).
+
+## Figure preparation
+
+Figures were prepared using UCSF ChimeraX <sup>46</sup>, CueMol (http://www.cuemol.org). To quantify binding ratio and DNA cleavage efficiency, we used ImageJ and GraphPad Prism. Both binding ratio and DNA cleavage efficiency were estimated. To quantify the binding ratio of Cas9 to nucleosomes and the efficiency of nucleosomal DNA cleavage, we quantified the bands in Native-PAGE using ImageJ. We quantified the fluorescence of Cy3 in H4 in the gel for the binding ratio, and 6-FAM at the 5’-end of nucleosomal DNA for the DNA cleavage efficiency. However, since it was difficult to label tail-less nucleosomes with fluorescence, we quantified the fluorescence of Cy5 in Cas9 for the binding rate and the band of EtBr-stained DNA for the DNA cleavage efficiency. The quantified data was imaged by GraphPad Prism. The binding ratio was calculated as the ratio of the intensity of the band indicating the complex to the sum of the intensity of the bands indicating the complex and the nucleosome. For calculating DNA cleavage efficiency, the difference between the intensity of the band nucleosomes were loaded and the the band Cas9 added was divided by the intensity of the band only nucleosomes loaded.
+
+## Transformation of CRISPR/Cas9 vector into rice callus
+
+Binary vectors containing three sgRNAs, rice codon-optimized SpCas9-WT or -H1264A/R1298Q, H1264D/R1298Q, H1264E/R1298Q, and hygromycin phosphotransfease (HPT) expression cassettes (Fig. 4d and Supplementary Table 2) were transformed into rice calli by *Agrobacterium*. *Agrobacterium*-mediated transformation of rice (*Oryza sativa* L. cv. Nipponbare) using scutellum-derived calli, as described previously <sup>47</sup>. Briefly, 1-month-old cultured rice calli were infected with *Agrobacterium* [strain EHA105 <sup>48</sup>]. After 3 days of co-cultivation, the calli were transferred to callus induction medium containing 50 mg/l hygromycin B (Wako Pure Chemicals) and 25 mg/l meropenem (Wako Pure Chemicals). Hygromycin-resistant calli were selected over 4 weeks and DNA was extracted from 24 independent calli for each transformation as described previously <sup>49</sup>.
+
+## Amplicon sequence analysis
+
+The first PCR was conducted with the primer sets listed in Supplementary Table 2. PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, CA, USA) and used as templates for a 2nd round of PCR to attach sequence adapters (IDT for Illumina DNA/RNA UD indexes, Illumina, CA, USA) for amplicon-sequencing libraries. The second round PCR products were purified using Agencourt AMPure XP beads. The fragment sizes were checked by agarose gel electrophoresis, and the concentration were measured using a Qubit 2.0 Fluorometer and a Qubit dsDNA HS Assay Kits (ThermoFisher Scientific). The sequencing libraries were subjected to paired-end sequencing on a Miseq sequencer (Illumina). Amplicon sequence results were analyzed using CRISPResso2 (http://crispresso2.pinellolab.org) <sup>50</sup>.
+
+## Statistical analysis
+
+Data are presented as means ± s.d. The alpha level was 0.05. Statistical analyses were performed in GraphPad Prism, using t tests as indicated.
+
+# References
+
+1. Hille, F. et al. The Biology of CRISPR-Cas: Backward and Forward. Cell 172, 1239–1259 (2018).
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+18. Hinz, J. M., Laughery, M. F. & Wyrick, J. J. Nucleosomes Selectively Inhibit Cas9 Off-target Activity at a Site Located at the Nucleosome Edge. J. Biol. Chem. 291, 24851–24856 (2016).
+19. Makasheva, K. et al. Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor. J. Am. Chem. Soc. 143, 16313–16319 (2021).
+20. Handelmann, C. R., Tsompana, M., Samudrala, R. & Buck, M. J. The impact of nucleosome structure on CRISPR/Cas9 fidelity. Nucleic Acids Res. (2023) doi:10.1093/nar/gkad021.
+21. Li, G. & Widom, J. Nucleosomes facilitate their own invasion. Nat. Struct. Mol. Biol. 11, 763–769 (2004).
+22. Bilokapic, S., Strauss, M. & Halic, M. Histone octamer rearranges to adapt to DNA unwrapping. Nat. Struct. Mol. Biol. 25, 101–108 (2018).
+23. Zuo, Z. & Liu, J. Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage. Curr. Opin. Struct. Biol. 62, 166–174 (2020).
+24. Farnung, L., Vos, S. M., Wigge, C. & Cramer, P. Nucleosome-Chd1 structure and implications for chromatin remodelling. Nature 550, 539–542 (2017).
+25. Kujirai, T. et al. Methods for Preparing Nucleosomes Containing Histone Variants. Methods Mol. Biol. 1832, 3–20 (2018).
+26. Han, Y., Reyes, A. A., Malik, S. & He, Y. Cryo-EM structure of SWI/SNF complex bound to a nucleosome. Nature 579, 452–455 (2020).
+27. Shibata, M. et al. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy. Nat. Commun. 8, 1430 (2017).
+28. Chen, F. et al. Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting. Nat. Commun. 8, 14958 (2017).
+29. Li, M. & Wang, M. D. Chapter Two - Unzipping Single DNA Molecules to Study Nucleosome Structure and Dynamics. in Methods in Enzymology (eds. Wu, C. & Allis, C. D.) vol. 513 29–58 (Academic Press, 2012).
+30. Ngo, T. T. M., Zhang, Q., Zhou, R., Yodh, J. G. & Ha, T. Asymmetric unwrapping of nucleosomes under tension directed by DNA local flexibility. Cell 160, 1135–1144 (2015).
+31. Mauney, A. W., Tokuda, J. M., Gloss, L. M., Gonzalez, O. & Pollack, L. Local DNA Sequence Controls Asymmetry of DNA Unwrapping from Nucleosome Core Particles. Biophys. J. 115, 773–781 (2018).
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+33. Tóth, K. et al. Histone- and DNA sequence-dependent stability of nucleosomes studied by single-pair FRET. Cytometry A 83, 839–846 (2013).
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+36. Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).
+37. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
+38. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
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+40. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
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+47. Toki, S. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol. Biol. Rep. 15, 16–21 (1997).
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+49. Edwards, K., Johnstone, C. & Thompson, C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 19, 1349 (1991).
+50. Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
+
+# Supplementary Files
+
+- [ExtendedDataFigureLegends.docx](https://assets-eu.researchsquare.com/files/rs-5031908/v1/3913d02384e3b5e2cf7c8de1.docx)
+- [CasNucpaperFigSup.pdf](https://assets-eu.researchsquare.com/files/rs-5031908/v1/b193bc0d9c61ac17f0d6ca41.pdf)

07fe21ef0f6d474fe8b7e0dc7cd070db30935ce26b44907931b3dc3bbd5ba6e5/preprint/images_list.json

@@ -0,0 +1,34 @@
+[
+    {
+        "type": "image",
+        "img_path": "images/Figure_1.png",
+        "caption": "Methodologies of metal oxide hydrogenations. (a) Schematic illustration of metal oxide hydrogenations via galvanic redox reaction between metal oxide and metallic cations. (b) Linear sweep voltammetry (LSV) curves for proton intercalation at pristine \u03b1-MoO3 under 1M H2SO4 solutions; two typical reduction reactions result in the orthorhombic H-doped MoO3 (HxMoO3), followed by the phase transition to the monoclinic HyMoO3 and (c) cyclic voltammetry (CV) curves of Pt electrode under Mo(\u2163) cation solutions, showing around 0.3 V vs. RHE of standard reduction potential. (d) Portion of the Pourbaix diagram for molybdenum oxide, Cu metal, and redox couples for molybdenum and vanadium ions under acidic conditions.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_2.png",
+        "caption": "Material characterization of pristine and partially reduced H-doped MoO3 (HxMoO3, x<0.4). (a) X-ray diffraction (XRD) analysis of pristine and partially reduced MoO3. The reduced MoO3 samples indicate that the strong diffraction patterns for the (020), (040) and (060) of MoO3 shift to lower angles, reflecting the wider van der Waals gaps, along with the different concentration of Mo(\u2163). X-ray photoelectron spectroscopy (XPS) analysis of pristine and reduced MoO3 in the (b) Mo 3d and (c) O 1s regions. Mo 3d spectra shows the gradually increasing Mo5+ oxidation states. (d) Fourier transform infrared spectroscopy (FTIR) analysis and (e) structure configuration of pristine MoO3 and H-MoO3 calculated by DFT.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_3.png",
+        "caption": "Electrochemical properties of pristine MoO3 and partially reduced H0.39MoO3 (H-MoO3). (a) Comparison of the third CV curves of pristine and H-MoO3, measured between 1.5 and 3.5 V vs. Li/Li+ at 0.1 mV s-1 of scan rate. (b) Voltammetric charge/discharge plateau of H-MoO3 measured at various potential sweep rates from 0.1 to 1.0 mV s-1. (c) The log (scan rate) versus log (peak current density) plots for major cathodic peak with b-values as indicator to compare the pseudo-capacitive properties of pristine MoO3 and H-MoO3. (d) Rate capability at different current from 100 to 3,000 mA g-1, followed by (e) cycling stability tests of MoO3 and H-MoO3 measured at a 1 A g-1.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    },
+    {
+        "type": "image",
+        "img_path": "images/Figure_4.png",
+        "caption": "DFT calculation results. Calculated formation energies corresponding to Li-ion interposition in (a) bulk \u03b1-MoO3 and (b) H-MoO3, * Mark represents the lithiation sites derived by proton interferences inducing lattice disorder. (c) Calculated energy barriers for Li-ion diffusion along the Li-ion accommodatable sites and (d) schematic illustration of feasible Li-ion diffusion pathways through the H-MoO3 lattice.",
+        "footnote": [],
+        "bbox": [],
+        "page_idx": -1
+    }
+]

07fe21ef0f6d474fe8b7e0dc7cd070db30935ce26b44907931b3dc3bbd5ba6e5/preprint/preprint.md

@@ -0,0 +1,108 @@
+# Abstract
+
+Rational reforming of metal oxide has a potential importance to modulate their inherent properties toward appealing characteristics for various applications. Here, we present a detailed fundamental study of the proton migration phenomena between mediums and propose the methodology for an exquisitely controllable metal oxide hydrogenation through galvanic reactions with metallic cation under ambient atmosphere. As a proof of concept for hydrogenation, we study the role of proton adoption on the structural properties of molybdenum trioxide, as a representative, and its impact on redox characteristics in Li-ion battery (LiB) systems using electrochemical experiments and first-principles calculation. The proton adoption contributes to a lattice rearrangement facilitating the faster Li-ion diffusion along the selected layered and mediates the diffusion pathway that promote the enhancements of high rate performance and cyclic stability. Our work provides great physicochemical insights of hydrogenations and underscores the viable approach for improving the redox characteristics of layered oxide materials.
+
+Physical sciences/Materials science/Materials for energy and catalysis/Corrosion  
+Physical sciences/Materials science/Materials for energy and catalysis/Batteries  
+Physical sciences/Materials science/Condensed-matter physics/Electronic properties and materials
+
+# Introduction
+
+In the research field of materials engineering, hydrogenating is one of the promising methods to manipulate the electrical and redox characteristics of metal oxides towards the favorable features for diverse applications<sup>1-5</sup>. The rearrangements of the crystal lattice with the deviation of metal atoms from their equivalent states have enormous impacts on the dielectric properties and energy states of electron of metal oxide during proton insertion, and therefore, a lot of effort has been devoted to tunable hydrogenation for these purposes. Moreover, inspired by proton migration into the oxides, there has been significant interest in oxide hydrogenation as a feasible strategy of hydrogen energy transportation for upcoming hydrogen economy<sup>6-8</sup>. Furthermore, recent studies have emphasized the vital role of H-binding energy of supported oxides to enhance the catalytic activities of primary catalyst in electrolysis and photo-electrochemical systems<sup>9-13</sup>.
+
+It has long been known that proton doping significantly impacts the inherent characteristics of metal oxide<sup>13,14</sup>. Therefore, the elucidation of the proton diffusion phenomenon from a hydrogen-rich atmosphere to the surface of hydrogen-poor metal oxides is important for further developments. To address this challenge, many efforts have been devoted to revealing this behavior. Experiments have verified that the sacrificial metals that have lower work functions accelerate the transfer of electron-proton pairs into the oxide lattice, motivating work on tunable oxide hydrogenation using sacrificial solid metal<sup>4,5,15,16</sup>. With respect to the hydrogenating phenomena itself, an exploration of proton movements using first-principles calculation revealed that the negatively charged oxide surface, driven by electron accumulation, contributes to lower energy barriers for proton migration<sup>15,16</sup>. Moreover, it has been confirmed that the proton capable metal oxide exhibits various phase conditions depending on a degree of the hydrogenation<sup>17,18</sup>, unlocking the great opportunity of tunable hydrogenated oxide and selectivity for favorable features.
+
+However, it is worth mentioning that the question about natural forces governing the hydrogenation of the metal oxides has not been fully elucidated. Specifically, there are no significant clues or criteria to correlate hydrogenation origins with the degree of hydrogenation and the crystallographic order of lattice oxide. Undeniably, there have been many practical achievements, however, previous hydrogenation studies are often neglect to consider the regularity of various crystal phases, obscuring the conception of oxide hydrogenation. That is, due to the lack of the cornerstone correlating the fundamental origins and material characteristics, consistent explanations of the natural force covering the proton movement are overlooked; and therefore, the sole descriptor for the impact of H-binding on physicochemical characteristics of metal oxide still remained elusive.
+
+In this context, we present a detailed study of the oxide hydrogenation by devising a novel cation-solution treatment method for controllable hydrogenation in metal oxides. The basis of the original hypothesis was that the impetus of hydrogenation is likely to be associated with the equivalent electrochemical potentials difference between cation as a reducing agent and metal oxide. We attributed the proton migratory phenomena to the mixed potential-induced electrochemical galvanic reaction, which occurs at the interfaces of solid oxide and dissolved cations, accompanying with the proton migration from the acid reservoir, as depicted in <strong>Fig.</strong> <strong>1a</strong>. Moreover, to reveal the sole impact of H-binding on inherent electrochemistry of metal oxide, we have studied the electrochemistry of orthorhombic molybdenum trioxide (α-MoO<sub>3</sub>), one of the layered oxides having accommodatable sites for protons, and its hydrogenated state having identical orthorhombic crystal. In particular, the redox behavior was investigated in organic electrolyte based-lithium ion battery (LiB) system, which is a suitable platform for understanding the inserted proton effects while precluding proton intervention from the electrolyte. Remarkably, the redox characteristics of hydrogenated α-MoO<sub>3</sub> (H-MoO<sub>3</sub>) in LiB systems were verified to be totally determined by means of the proton doping along with the enhancements of high rate performance and cyclic stability. Typically, it is found that there was no capacity limitation for partially reduced H-MoO<sub>3</sub> in spite of the lower oxidation state of molybdenum at the expense of partial reduction with cation dopant. To elucidate this contradictory question, the completely distinguishable redox natures of H-MoO<sub>3</sub> were demonstrated by computational density functional theory (DFT) and the nudged elastic band (NEB) approach. It is especially worthy noticing that proton adoption and its impacts on an intermolecular interaction contribute to the advent of the new sites and pathway for lithium-ion diffusion.
+
+# Results
+
+Methodologies of galvanic redox reaction inducing metal oxide hydrogenations. Figure 1a illustrates a novel cation-solution treatment method that we designed for controllable hydrogenation in metal oxides. The linear sweep voltammetry (LSV) curves (Fig. 1b and Fig. S1a) indicate two distinct reductive potentials of 0.44 and 0.15 V SHE, respectively: first reduction peak corresponds to the proton and charge transport to α-MoO₃ (HₓMoO₃, x < 0.4), and the second peak indicates an additional reduction reaction for proton storage with phase transition into monoclinic phase (HᵧMoO₃, y > 0.4)¹⁷,¹⁸. The basic electrochemistry of the α-MoO₃ capacitive nature provides notable feature of oxide hydrogenation; and we focused here on the possibility of tunable hydrogenation strategy via galvanic reaction using specific ionic reductants having proper standard potential.
+
+To demonstrate that the mixed potential-induced galvanic reaction is the determinant of electron-proton pair migration, we studied the feasibility of Mo(IV) cation solution as hydrogenation trigger. The cyclic voltammetry (CV) analysis of Pt electrode under Mo(IV) solution, as shown in Fig. 1c and Fig. S1b, clearly shows that the oxidation current (for MoO²⁺(IV) + H₂O → MoO₂⁺(V) + 2H⁺ + 2e⁻) occurs at around 0.30 V SHE, which is in agreement with the Pourbaix diagram for molybdenum¹⁹. As a proof of concept for electrochemical galvanic reaction, we organized the standard potentials of various molybdenum oxide phases corresponding to the degree of hydrogenation (MoO₃/HₓMoO₃/HᵧMoO₃), and that of cations (Mo(IV) and V(II)), and solid Cu metal as well to reconsider the conventional solid-metal treatments method in the electrochemical point of view as shown in Fig. 1d. Firstly, we used dark-brownish Mo(IV) cation solution as reductant (the detailed explanation for MoO₃ modification method is described in Material preparation section and Fig. S2). The prepared hydrogenated α-MoO₃ using specific Mo(IV) concentration shows the two distinct reflection (XRD) patterns (Fig. 2a); major reflection peaks of pristine α-MoO₃ shift to lower angles by means of hydrogenation, representing a widened van der Waals (vdW) inter-layer gaps from 14.0 to 14.4 Å. Additionally, the X-ray photoelectron spectroscopy (XPS) analysis confirmed that Mo⁵⁺ species in Mo 3d spectra (Fig. 2b) gradually and selectively increases with the occurrence of the surface adsorbed species corresponding to –OH in O 1s spectra (Fig. 2c) accompanying with chemical shifts of lattice oxygen to lower binding energy. A stoichiometry of H-MoO₃, calculated by area ratio of Mo⁵⁺ and Mo⁶⁺ in Mo 3d spectra (Fig. 2b), is determined to be H₀.₃₉₂MoO₃, which agrees with the electrochemical results for H-doped α-MoO₃ (HₓMoO₃, x < 0.4). Whereas, in case of cation-solution treatments using violet-colored V(II) solution as hydrogenation trigger, whose reduction potential value (V(III)/V(II), -0.255 V SHE)²⁰ is lower than that of HᵧMoO₃/HₓMoO₃ (x < 0.4 < y, 0.15 V SHE), the V(II) cation treated α-MoO₃ shows the monoclinic phase as shown in Fig. S3. In this case, XPS results show a varying oxidation state in Mo 3d spectra with increased surface absorbed species (Fig. S4), implying the presences of an additional redox reaction with proton insertion compared with the Mo(IV) treated case (HₓMoO₃, x = 0.392). We also examined the feasibility of cation-treatment methods using commercial WO₃ (c-WO���, monoclinic), whose reduction potential for WO₃/HₓWO₃ is lower than 0.3 V SHE²¹, and verify the identical trend of oxide hydrogenation (Figs. S5 and S6). In this regard, metal oxide hydrogenation strategy using solid state metal also could be accepted since the reduction potential of Cu metal (Cu/Cu²⁺, 0.34 V SHE, Fig. 1d) is lower than that of molybdenum oxide (MoO₃/HₓMoO₃, 0.44 V SHE) even though this method requires for extra efforts to eliminate the solid metal residual. Hereby, these results provide the reasonable deduction to unveil the clues of the hydrogenation origins. Hence, considering the restrictive crystal phase and the basic electrochemistry of proton capable oxide in acidic condition by means of hydrogenation degree, oxide hydrogenation could be driven spontaneously by galvanic reaction via cation-solution treatment; namely, the primary keystone of controllable oxide hydrogenation determining the extent of proton doping and crystal phases is the standard potential differences between oxide material and metallic cation.
+
+The next question in our study was oriented toward the physicochemical properties of H-MoO₃ since there was no significant variation in morphology, crystallographic plane, and atomic vibrational Raman spectra as shown in Figs. S7 and S8. Thereby, it is the optimum conditions to investigate the sole impact of H-binding. Whereas, contrary to crystallographic analysis results, Fig. 2d shows the distinct features in FTIR vibration signals of H-MoO₃, especially the absence of stretching vibration frequency corresponding to the Mo-Oₐ (asymmetric oxygen) bond represented at 827.4 and 819.0 cm⁻¹. Instead, a noticeable peak at 627.4 cm⁻¹ is observed after proton doping. We used DFT calculation to estimate the stabilized H-sites of H-MoO₃ (Fig. S9), through which it is found that intersectional asymmetric oxygen sites are the energetically favorable for proton dangling as depicted in Fig. 2e. Notably, it is extraordinary feature of proton doped α-MoO₃ since cation doping strategies with large dopant radius are typical approaches to increase the vdW gaps via pre-intercalation in inter-layer spaces. Considering the relative radius of cations including Li⁺ (0.90 Å), Na⁺ (1.16 Å), and K⁺ (1.52 Å) or molecule such as H₂O (vdW radius, 1.70 Å)²²–²⁴, it is reasonable to consider that these dopants would be incorporated in vdW layer (14.0 Å for pristine MoO₃) surrounded by terminal oxygen rather than inner-plane sites. In contrast, protons, having the lowest occupation of 0.87 × 10⁻⁵ Å, are more likely to be located at neighboring lattice oxygen atoms of octahedral MoO₆ (Fig. 2e). Thus, the triggers of the noticeable lattice distortion are entirely distinguishable, raising a different perspective of varying characteristic of metal oxide by means of dopant types and sites.
+
+Electrochemical properties of H-doped orthorhombic MoO₃. To elucidate the effects of the H-binding on electrochemical characteristics of α-MoO₃, electrochemical analyses in LiB systems for pristine MoO₃ and H-MoO₃ (HₓMoO₃, x = 0.392) were conducted as shown in Fig. 3. Specifically, the orthorhombic structure of α-MoO₃ takes an accommodatable sites for Li-ion; inner-plane (intra-layer site) of MoO₆ adjacent with Oₐ and Oₛ (symmetric oxygen) and inter-plane (inter-layer site) of vdW gaps neighboring Oₜ (terminal oxygen) lattices of MoO₃ (Fig. 2e)²⁵. However, there is a pervasive problem of irreversible phase transition reaction at pristine state of α-MoO₃ shown in reduction peaks for Li-ion intercalation at 2.70 vs. Li/Li⁺ (Fig. S10a)²⁴,²⁵. In contrast, the initial CV curve for H-MoO₃ indicates a suppressed current plateau around 2.70 V vs. Li/Li⁺ and an obvious reduction peak at 2.38 V vs. Li/Li⁺ as shown in Fig. S10b. For further clarification on this irreversibility, ex-situ XRD analysis of (de)lithiated electrodes were conducted. In Fig. S11, ambiguous XRD patterns are observed for both lithiated MoO₃ and H-MoO₃, whereas, completely different results are seen following delithiation. There are uncertain diffraction peaks as a result of the irreversible phase transition for delithiated MoO₃ during the initial cycle; in contrast, H-MoO₃ shows the well-maintained crystallographic order of orthorhombic structure. It is remarkable that Mo-Oₐ bonding changes with lattice rearrangements would prohibit the irreversible Li-ion intercalation at the intra-layer sites of MoO₆.
+
+After the 2nd activation cycle, H-MoO₃ achieves a reversible specific capacity of 1009.4 C g⁻¹ (280.4 mAh g⁻¹) at 0.1 mV s⁻¹, which is higher than the capacity realized for pristine MoO₃ (946.8 C g⁻¹, 263 mAh g⁻¹) and close to the theoretical capacity (1005 C g⁻¹, 279 mAh g⁻¹)²⁵, as shown in Fig. 3a. Notably, it exhibits an additional pair of redox potential at 2.95 and 2.68 V vs. Li/Li⁺ for H-MoO₃, and that is in stark contrast to the original redox characteristics in a whole range of high scan rates as shown in Fig. 3b. Besides, in quantitative capacitive analysis of the Li-ion intercalation behaviors, the b-value corresponding to major lithiation of pristine MoO₃ was 0.71, whereas that of H-MoO₃ was 0.88 as shown in Fig. 3c, representing the enhanced pseudo-capacitive like Li-ion diffusion features by the proton introduction²⁶,²⁷ (detailed instructions for correlation between sweep rate, redox peak current density, and b-value as the indicator of capacitive ion diffusion are described in Fig. S12). Similarly, log (ʋ) versus log (i) plot for anodic peak current (Fig. S12d) also indicates the enhanced capacitive Li-ion diffusion features of H-MoO₃. This series of enhanced capacitive characteristics are also represented by enhanced rate capability shown in Fig. 3d. Typically, at the current of 1.0 A g⁻¹, H-MoO₃ achieved a stable specific capacity of 170.8 mAh g⁻¹, while pristine MoO₃ has 136.0 mAh g⁻¹ with a small decline in capacity. Thereafter, as shown in Fig. 3e, pristine MoO₃ shows the rapid capacity decaying early in the cycling process, capacity decrease to 84.1 mAh g⁻¹ after 250 cycles, whereas, H-MoO₃ shows the remarkably enhanced cycling stability, exhibiting 84.7% (144.3 mAh g⁻¹) and 72.1% (122.9 mAh g⁻¹) of capacitance retention at 1.0 A g⁻¹ during 500 and 1,000 cycles, respectively.
+
+We further take interest in the existence of multiple redox peaks, and especially in an enhanced capability of H-MoO₃. An intuitive advantage of the H-binding introduction has been known as band-gap tuning by advent of H-doping level²⁸,²⁹, which could be confirmed by our DOS calculations (Fig. S13). Then, to elucidate the redox characteristics, the formation energies for lithiated state of both pristine MoO₃ and H-MoO₃ were calculated. The pristine state retains the two accommodatable sites for Li-ion (inter/intra site) having formation energy of -2.023/-1.648 eV (Fig. 4a, Fig. S14); as discussed, however, due to the irreversible lithiation at the intra-layer site during initial discharge process, the redox peak of pristine state shows the one reversible major peak corresponding to the lithiation at the inter-layer sites. Whereas, since the H-MoO₃ exhibits the asymmetrical distribution of proton insertion as a consequence of limited proton adoption (Fig. 2e), the formation energies for inter and intra-layer sites are divided into two groups as shown in Fig. 4b (Fig. S15, * marks imply the lithiation sites derived from the lattice asymmetry): that is, the asymmetric lattice order induced by limited hydrogenation would result in differences of formation energy and distinctive redox potential (Fig. 3a). Moreover, first-principle calculation results in Fig. S15 indicate that the proton adoption sites are distinguished from the Li-ion accommodatable sites, through which it is confirmed that H-binding of H-MoO₃ would have less influences on preservable Li-ion sites, facilitating a comparable capacity realization with pristine state. Thereby, even though it has long been accepted that there is a trade-off in relation to cation doping with entailed capacity limitation²²,²³,²⁵, this assertion contains a principal exception in case of H-MoO₃ since the lithium intercalation sites are rearranged not by the cation interposition but by proton interference on Mo-Oₐ bonding.
+
+We also investigated a role of H-binding in determining the diffusion rates by constructing the Li-ion diffusion paths and calculating the energy barriers using NEB simulation. According to the ARXPS results (Fig. S16), showing that the proton doping would occur throughout the surface and bulk regions of metal oxide, the energy barriers for available diffusion pathways of both pristine MoO₃ and H-MoO₃ (Figs. S17 and S18) were calculated on the basis of the most stable inter-layer site. The energy barrier for single Li-ion diffusion pathway along the inter-layer sites in pristine MoO₃ is 0.5 eV, whereas, in case of H-MoO₃ having various diffusion routes due to the asymmetrical lattice orders, the rate determining energy barrier for Li-ion moving along the inter-layer sites is only 0.397 eV as shown in Fig. 4c. Moreover, the energy barrier of Li-ion moving from intra-layer to inter-layer is 0.338 eV as shown in Fig. S18. That is, through the advent of inter-layer* position with various diffusion pathways as a result of asymmetrical lattice order by proton introduction, the bottleneck energy barrier for Li-ion diffusion through the bulk regions could be lowered by approximately 0.1 eV as shown in Fig. 4d. Therefore, we concluded that lattice distortion by proton adoption would modulate the electrical and redox characteristics, resulting in reversible cycling performance and faster kinetics of Li-ion along the intercalation sites of H-MoO₃.
+
+# Discussion
+
+In this work, we established the cornerstone of metal oxide hydrogenation by devising a novel cation-solution treatment strategy and demonstrated that the impetus of a proton diffusion through the oxide lattice is based on the galvanic redox reactions. Herein, we could verify though the experimental proof that the standard potentials of oxide and metallic cations are vital factors for the controllable proton doping. This methodology not only encompasses the implicit principle of conventional solid-metal treatment for oxide hydrogenation but also unveils the phase transition mechanism of metal oxide during electron-proton pair migration. Based on the thorough understanding of hydrogenation mechanisms, sole impact of H-binding with practical applicability of this method for intercalation-type energy storage materials in lithium-ion battery systems are demonstrated. In battery systems, there are significant differences in redox characteristics as protons participate in orthorhombic-MoO₃. Typically, the proton doping with lattice rearrangement would cause the suppressed irreversible phase transition reaction and accelerated Li-ion diffusion with enhanced charge transfer reaction. Through the first principle DFT calculation, it is confirmed that the interposition of protons was entirely distinguished compared with other cation dopants, which results suggest the new possibilities to overcome the trade-off in relation between cation doping and limited capacity for energy storage. Moreover, it could be demonstrated via NEB approach that asymmetric lattice order results in diffusion pathways of Li-ion having lower energy barriers along the intercalation sites, contributing to enhanced diffusion rates and pseudo-capacitive characteristics. We believed that this work would suggest new insight of oxide reforming strategy via corrosion-based galvanic reaction and highlight the sole impact of H-binding on inherent properties of oxide for preferable materials engineering.
+
+# Methods
+
+## Material preparation.
+
+Orthorhombic structure MoO₃ (α-MoO₃) was synthesized through a one-step hydrothermal synthesis. Ammonium molybdate tetrahydrate (2 g, Sigma Aldrich) was added to 30 ml of deionized water and 10 ml of nitric acid (60%, Daejung) to form a solution. The prepared solution was transferred into a 100 ml Teflon-lined autoclave and heated in box furnace at 180 °C with the constant 5 °C min⁻¹ of heating rate for 12 h. The obtained white powder was washed with distilled water several times using vacuum filtering, and dried under vacuum at 70 °C. As for monoclinic WO₃, it is purchased from Sigma-Aldrich (CAS no: 1314-35-8) and used without any purifications. For metal oxide hydrogenation, Molybdenum (Ⅳ) solution was prepared by dissolving the specific amount of molybdenum chloride (MoCl₅, Sigma Aldrich) in 10 ml of acetonitrile (CARLO ERBA) and 10 ml deionized water. Vanadium (Ⅱ) solution was prepared by dissolving the specific amount of ammonium metavanadate dissolved in 30 ml of deionized water and 10 ml of sulfuric acid (Daejung) and excess amount of zinc metal powder was added. The obtained solution was refined using vacuum filtering to remove the residual metal. Galvanic redox reaction induced metal oxide hydrogenation begin as the metallic cation solutions are added at the 0.2 g of metal oxide (MoO₃, WO₃) dispersed deionized water and stirred for 3 h at room temperature. The reduced metal oxide powder was washed with deionized water for several times to remove the residual metallic cation solutions and collected using vacuum filtering.
+
+## Material characterization.
+
+The structural properties of samples were analyzed by X-ray diffraction (XRD; Rigaku) using Cu Kα radiation (λ = 0.15406 nm) and the interlayer plane distances were estimated by the using (020) reflection. Ex-situ XRD was performed in Rigaku (Smartlab) using Cu Kα radiation (λ = 0.15406 nm) by film mode. The samples were prepared after electrode polarization by linear sweep voltammetry (LSV) with the 0.1 mV s⁻¹ of scan rate in Li-ion battery coin-cell systems. The electrode potential shift to 1.5 and 3.5 V vs. Li/Li⁺, respectively, to evaluate the fully charged/discharged condition, and the polarized electrodes were obtained after cell disassembling, followed by rinsing with dimethyl carbonate (DMC) solvent and dried naturally in argon purged glove box. Oxidation configurations of prepared samples are conducted using X-ray photoelectron spectroscopy (XPS, Thermo Fisher Scientific) using Al Kα (1486.6 ev) for excitation source at 100 W, and angle-resolved XPS (ARXPS) analysis for H-MoO₃ was conducted by film mode with the photoelectron tilt angle from zero to 70 °. The surface morphologies of synthesized MoO₃ were collected using high resolution transmission electron microscopy (TEM, Tecnain Fe F30 S-Win) and high-resolution scanning electron microscopy (SEM, S-4700 with EMAX system, Hitachi), and the element mapping images of MoO₃ were measured by JSM-7500F (JEOL). Raman spectroscopy (Horiba) was measured with BX41 confocal microscope and the Fourier transform infrared spectrometer (FT-IR, Bruker) was measured by transmission mode.
+
+## Electrochemical analysis.
+
+The redox features of pristine MoO₃ under acidic condition were measured in a three-electrode systems using a rotating disk electrode (RDE, 0.196 cm², glassy carbon) as working electrode, saturated calomel electrode (SCE) as reference electrode, and Pt mesh as counter electrode. The polarization curve of pristine MoO₃ was measured by linear sweep voltammetry (LSV) with the 10 mV s⁻¹ of scan rate from open circuit voltage (OCV, around 0.4 V SCE) to -0.8 V SCE under N₂ saturated 1M H₂SO₄ solution. The standard redox potential of molybdenum cation under 1M H₂SO₄ was measured in a three-electrode system using Pt mesh, SCE, and Pt rod as working, reference, and counter electrode, respectively. The polarization curves were measured by cyclic voltammetry (CV) between −0.3 to 0.8 V SCE with the 10 mV s⁻¹ of scan rate. For the conversion of the applied potential vs. SCE, the potentials of the obtained results are converted to the reversible hydrogen electrode (RHE) according to Nernst Equation: E RHE = E SCE + E° SCE + 0.059 pH. The redox characterizations of prepared samples in Li-ion battery systems were investigated in two-electrode system using CR2032-type coin cell. The slurry, composed of the active materials (pristine MoO₃, H-MoO₃), supporting carbon (Super P), and binder (PAA) with a weight ratio of 7:2:1, was cast on Al foil, and the electrode was dried at 60 °C for 24 h in a vacuum oven. The coin cell was assembled with prepared electrode with a diameter of 10 mm (around 0.4 ~ 0.6 mg of loading mass: 0.50 ~ 0.76 mg cm⁻²) as working electrode, Li metal (16 pi) as counter/reference electrode, a piece of propylene film (Celgard 2400) as separator, and 1 M of lithium hexafluorophosphate (LiPF₆) dissolved in a mixture of ethylene carbonate and diethylene carbonate (EC/DEC, 1:1 by volume) as electrolyte in an argon gas-filled glove box. The electrochemical properties of assembled coin cell were investigated between 1.5 and 3.5 V vs. Li/Li⁺ of potential regions using potentiostat (Solatron Analytical, 1470E/1400 CellTest System) and battery cycler (WonATech, WBCS3000). The cyclic performances were measured at various specific current density of 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2.0, and 3.0 A g⁻¹ to evaluate the rate capability. Long-term cycling tests were performed at specific current density of 1,000 mA g⁻¹ for durability test in the potential range of 1.5–3.5 V vs. Li/Li⁺.
+
+## Computational details
+
+To investigate the energetics of Li ion migration within pristine MoO₃ and HₙMoO₃ with n = 0.5, we carried out first-principles electronic structure calculations within density functional theory (DFT) scheme as is implemented with Vienna Ab initio Simulation Package (VASP) 5.4. A plane-wave basis set with 500 eV energy cut-off is employed for the solution of the Kohn-Sham equation, and the exchange-correlation interactions among electrons are described within generalized gradient approximations (GGA). Moreover, the DFT-D3 functional of Grimme et al. is chosen to properly treat the van der Waals interactions in MoO₃ and H₀.₅MoO₃ (Table S1). To optimize all geometric structures, the unit cells of pristine MoO₃ and H₀.₅MoO₃ which is two times larger in the y-direction, due to the setting for n = 0.5, are fully relaxed until the energy and force differences between successive relaxation steps becomes less than 10⁻⁵ eV and 10⁻² eV/Å, respectively. In calculations, 3×2×1 and 3×1×1 supercells are employed for MoO₃ and H₀.₅MoO₃, respectively, together with Γ-centered 3×5×3 k-point grids for the Brillouin zone sampling.
+
+To study Li diffusion, the energy barrier simulations are performed based on the climbing image nudged elastic band (CI-NEB) method with a force-based optimization scheme. The forces convergence threshold orthogonal to the band is set to be less than 0.01 eV/Å in our CI-NEB calculations. For evaluating the structural stability of the initial and final states, formation energies (ΔHf) are calculated (Table S2) by the following equations:
+
+$$
+\Delta H_f = E[\text{Li} + (\text{H}_{0.5})\text{MoO}_3] - E[(\text{H}_{0.5})\text{MoO}_3] - E[\text{Li}]
+$$
+
+where the E[Li + (H₀.₅)MoO₃] and E[(H₀.₅)MoO₃] are the total energy of (H₀.₅)MoO₃ system with and without one Li atom, respectively, and E[Li] is the chemical potential which is the per atom energy from bcc Li metal.
+
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+
+# Supplementary Files
+
+- [SupplementaryInformation.docx](https://assets-eu.researchsquare.com/files/rs-3998371/v1/8961de55798ac2b942a8b36f.docx)