Continuous-wave Electron Paramagnetic Resonance Spectra Research Articles

Continuous-wave and pulsed EPR studies of glass-like carbon with high spin concentration: Evidence for triplet states

Glass-like carbons (GLCs) are a large group of disordered carbon materials which have applications in various fields, but not all their properties are well characterized. In particular, they have high concentration of paramagnetic centers (PCs) which are easily observed by electron paramagnetic resonance (EPR). However, an apparently simple EPR spectrum, together with its peculiarities caused by high electrical conductivity, make it difficult to identify the PCs and their origin remains unknown. To resolve these unsettled questions, in this work we use the vacuum Rabi splitting and Rabi oscillations which so far have not been applied to study these materials by the continuous wave (CW) and pulsed EPR. GLC produced by pyrolysis of phenol-formaldehyde resin at 1120 K was studied in the temperature range of 4.3–300 K. Using the Rabi splitting we found conditions for undistorted registration of CW EPR spectra and determined spin concentration (1×1020 spin/g). The temperature dependence of the EPR susceptibility shows the coexistence of Pauli paramagnetism due to conduction electrons and the Curie-type paramagnetism of thermally activated PCs with the activation energy of 5.7 cm-1. These PCs may be excited triplet states, the presence of which is revealed by Rabi oscillations in the pulsed EPR.

Glass-like behavior of intercalated organic solvents in graphite oxide detected by spin-probe EPR.

Membranes based on graphite oxide (GO) are promising materials for the separation of polar liquids and gases. Understanding the properties of solvents immersed in GO is important for the development of various technological applications. Here, the molecular motions of the TEMPO nitroxide spin probe in acetonitrile intercalated into the GO inter-plane space were studied using electron paramagnetic resonance (EPR), including its pulsed version, and electron spin echo (ESE). For a sample containing 75% acetonitrile relative to equilibrium sorption at room temperature, ESE-detected stochastic librations were observed for TEMPO molecules above 135 K. Since these librations are an inherent property of molecular glasses, this fact indicates that intercalated acetonitrile forms a two-dimensional glass state. Above 225 K, an acceleration of stochastic librations was observed, indicating the manifestation of a glass-like dynamical cross-over. Continuous wave (CW) EPR spectra of TEMPO showed the absence of overall tumbling motions in the entire investigated temperature range of up to 340 K, indicating that the intercalated acetonitrile does not behave as a bulk liquid (the melting point of acetonitrile is 229 K). Dynamical librations of TEMPO molecules detected by CW EPR were found to accelerate above 240 K.

Influence of GeO2 Content on the Spectral and Radiation-Resistant Properties of Yb/Al/Ge Co-Doped Silica Fiber Core Glasses.

In this study, Yb/Al/Ge co-doped silica fiber core glasses with different GeO2 contents (0–6.03 mol%) were prepared using the sol–gel method combined with high-temperature sintering. The absorption, fluorescence, radiation-induced absorption, continuous-wave electron paramagnetic resonance spectra, and fluorescence decay curves were recorded and analyzed systematically before and after X-ray irradiation. The effects of GeO2 content on the valence variations of Yb3+/Yb2+ ions, spectral properties of Yb3+ ions, and radiation resistance of Yb/Al/Ge co-doped silica glasses were systematically studied. The results show that even if the GeO2 content of the sample is relatively low (0.62 mol%), it can inhibit the generation of Yb2+ ions with slight improvement in the spectral properties of Yb3+ ions in the pristine samples and effectively improve its radiation resistance. Direct evidence confirms that the generation of trapped-electron centers (Yb2+/Si-E’/Al-E’) and trapped-hole centers (Al-OHC) was effectively inhibited by Ge co-doping. This study provides a theoretical reference for the development of high-performance, radiation-r esistant Yb-doped silica fibers.

CW-EPR spectra of P1 centers in HPHT diamond in the vicinity of Rabi resonance: possible standard for [formula omitted] evaluation in EPR and DNP enhanced NMR spectroscopies

Synthetic commercial type-IIa diamond crystal containing low concentration of P1 (Ns0) centers ([Ns0] <1ppm) was examined as a possible calibration standard for evaluating microwave magnetic field intensity (B1) in the spectrometer cavity. The reliability of this standard was experimentally and theoretically tested by employing two different types of microwave cavities with different distributions of B1 values. The procedure relies on the ordinary continuous wave electron paramagnetic resonance (CW-EPR) with magnetic field modulation (ωrf/2π=100 kHz) applied under the regime of the microwave power (PMW) saturation of the out-of-phase first harmonic signal. The power saturation procedure for PMW∼B11/2 was analyzed with respect to the change of spectral lineshape when Rabi frequency (ω1=γB1, γis the electron gyromagnetic ratio) approaches to ωrf. The phenomenon of the inversion of sideband lines while passing the Rabi resonance condition (ω1=ωrf) was analyzed. In specific, from the simulation of the experimental lineshapes in the close vicinity of Rabi resonance (ω1≈ωrf) the correspondingB1 can be assigned. For the chosen diamond crystal low value of B1≈3.6µT at Rabi resonance was determined what makes it a convenient standard for DNP enhanced NMR spectroscopy which requires relatively small B1.

A Carbazole-Derived Nitroxide That Is an Analogue of Cytidine: A Rigid Spin Label for DNA and RNA.

A variety of semirigid and rigid spin labels comprise a valuable arsenal for measurements of biomolecular structures and dynamics by electron paramagnetic resonance (EPR) spectroscopy. Here, we report the synthesis and characterization of rigid spin labels Ċ and Ċm for DNA and RNA, respectively, that are carbazole-derived nitroxides and analogues of cytidine. Ċ and Ċm were converted to their phosphoramidites and used for their incorporation into oligonucleotides by solid-phase synthesis. Analysis of Ċ and Ċm by single-crystal X-ray crystallography verified their identity and showed little deviation from planarity of the nucleobase. Analysis of the continuous-wave (CW) EPR spectra of the spin-labeled DNA and RNA duplexes confirmed their incorporation into the nucleic acids and the line-shape was characteristic of rigid spin labels. Circular dichroism (CD) and thermal denaturation studies of the Ċ-labeled DNAs and Ċm-labeled RNAs indicated that the labels are nonperturbing of duplex structure.

Effect of the GeO2 content on the radiation resistance of Er3+ -doped silica glasses and fibers

In this work, Er/Al/Ge co-doped silica glasses with different GeO2 content (0–3 mol%), as well as Ge/Al co-doped silica glasses, are prepared by combining the sol–gel and high-temperature sintering methods. Further, the effects of the GeO2 content on the absorptions and emissions properties, and lifetimes of the glasses before and after 1KGy γ-ray irradiation are compared. The Er/Al/Ge co-doped silica fibers are prepared from a preform produced via modified chemical vapor deposition (MCVD) combined with nano sol-doping. The effects of Ge co-doping on the optical loss and amplifier gain of the Er-doped silica fibers (EDFs) before and after irradiation are also investigated. The related mechanism and species of the γ-ray radiation-induced color centers are revealed via radiation-induced-absorption (RIA) and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopies. The results revealed that co-doping with GeO2 considerably improves the radiation resistance of the glass and exerts a slight effect on the spectral properties of the Er/Al/Ge co-doped silica glasses before irradiation. The RIA and CW-EPR spectra revealed that the aluminum–oxygen hole center (AlOHC) defects reduce with increasing GeO2 content because the intermediaries, the Ge-related oxygen-deficient centers (GeODC(I) and GeODC(II)), exhibit stronger abilities to trap the holes compared with the [AlO4/2]− group. This reduces the RIA level in the visible and near-infrared regions of the Er/Al/Ge co-doped silica glass. The irradiation experiment on the fiber further confirmed that the radiation resistance of EDFs can be considerably improved by Ge co-doping.

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D2O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy.

Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein labeling; and utilizing Cu(II)NTA labeled proteins, to measure distance constraints and backbone dynamics. In doing so, EPR measurements are less influenced by sidechain motion, which influences the breadth of the measured distance distributions between two spins, as well as the measured residue-specific dynamics. More broadly, such EPR-based distance measurements provide unique structural constraints for integrative structural biophysics and complement traditional biophysical techniques, such as NMR, cryo-EM, FRET, and crystallography. Graphic abstract: Monitoring the success of Cu(II)NTA labeling.

193 nm excimer laser-induced color centers in Yb3+/Al3+/P5+-doped silica glasses

Yb3+, Yb3+/Al3+, and Yb3+/P5+ co-doped silica glasses, as well as pure silica, Al3+, and P5+ single-doped silica glasses, were prepared using a sol–gel method combined with high-temperature sintering. The glasses were irradiated with a 193 nm ArF excimer laser for different durations. The species of the 193 nm laser-induced color centers were identified by radiation-induced absorption (RIA), in-situ photoluminescence (PL), and continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopic techniques. To obtain the RIA and CW-EPR characteristics of each color center, the Gaussian decomposition of RIA and the line-shape simulation of the CW-EPR spectra were performed. The formation mechanism of the color centers was correlated with the glass microscopic structure, which was obtained by nuclear magnetic resonance (NMR) spectroscopy. The results show that the oxygen hole center and the Yb2+ ion pairs are primarily responsible for the radiation-induced darkening, and their formation is highly dependent on the charge balance between the Yb3+ ion and its ligand. This work provides insights into the underlying mechanism of pump light and/or external radiation-induced optical losses in Yb3+-doped silica fibers.

Enhanced radiation resistance of ytterbium-doped silica fiber by pretreating on a fiber preform

We propose a new pretreatment method on fiber preforms by loading deuterium (D2), pre-irradiation, combined with thermal annealing. The effect of the pretreatment condition on the optical loss at 1200 nm and a laser slope efficiency of ytterbium-doped silica fibers (YDFs) before and after γ-radiation was comparatively investigated. The related mechanism was revealed by combining the optical absorption, continuous wave electron paramagnetic resonance (CW-EPR), Raman, and Fourier transform infrared (FTIR) spectroscopies. A laser experiment shows that the radiation resistance of YDFs can be significantly improved by the pretreatment on a fiber preform. Also, this new pretreatment method has no obvious negative impact on the laser performance of non-irradiated YDFs. Furthermore, the vacuum experiment confirms that the YDF obtained by this method, named pretreated YDF, may have a long-term radiation stability when it is used in a vacuum environment (such as space). CW-EPR spectra show that the formation of color centers was effectively inhibited in pretreated YDF, which is correlated with the decrease of color center precursor and the existence of the deuterium radical as confirmed by Raman and FTIR spectra.

Electron paramagnetic resonance study of ferroelectric phase transition and dynamic effects in a Mn2+ doped [NH4][Zn(HCOO)3] hybrid formate framework.

We present an X- and Q-band continuous wave (CW) and pulse electron paramagnetic resonance (EPR) study of a manganese doped [NH4][Zn(HCOO)3] hybrid framework, which exhibits a ferroelectric structural phase transition at 190 K. The CW EPR spectra obtained at different temperatures exhibit clear changes at the phase transition temperature. This suggests a successful substitution of the Zn2+ ions by the paramagnetic Mn2+ centers, which is further confirmed by the pulse EPR and 1H ENDOR experiments. Spectral simulations of the CW EPR spectra are used to obtain the temperature dependence of the Mn2+ zero-field splitting, which indicates a gradual deformation of the MnO6 octahedra indicating a continuous character of the transition. The determined data allow us to extract the critical exponent of the order parameter (β = 0.12), which suggests a quasi two-dimensional ordering in [NH4][Zn(HCOO)3]. The experimental EPR results are supported by the density functional theory calculations of the zero-field splitting parameters. Relaxation time measurements of the Mn2+ centers indicate that the longitudinal relaxation is mainly driven by the optical phonons, which correspond to the vibrations of the metal-oxygen octahedra. The temperature behavior of the transverse relaxation indicates a dynamic process in the ordered ferroelectric phase.

UV absorption bands and its relevance to local structures of ytterbium ions in Yb3+/Al3+/P5+-doped silica glasses

Yb3+, Yb3+/Al3+, Yb3+/P5+, and Yb3+/Al3+/P5+ doped silica glasses were prepared via the sol-gel method combined with high-temperature sintering. The amorphous nature of as-prepared glasses was confirmed by the X-ray diffraction analysis. The composition dependent ultraviolet (UV) absorption changes in the glasses were correlated with the local structure of Yb3+ ions that was obtained by pulsed electron paramagnetic resonance (EPR). In situ photoluminescence evolution and continuous wave EPR spectrum of color centers induced by a 193-nm UV laser were recorded, and the role of UV absorption bands in the photodarkening process was discussed. The results indicated that the strong UV absorption bands in the range of 190–290 nm were primarily attributed to the charge-transfer (CT) transition from O2– to Yb3+ and the position of CT bands shifted to a higher energy level with increases in the electronegativity of next nearest neighbor atoms (Al/Si/P) of Yb3+ ions. Furthermore, the results revealed that excitation into the CT bands leads to the generation of oxygen hole centers and Yb2+ ions. The study is useful for understanding the underlying mechanism of photodarkening in Yb3+-doped high-power laser fibers.

Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

A systematic study on the self-assembled solution system of human serum albumin (HSA) and paramagnetic doxyl stearic acid (5-DSA and 16-DSA) ligands is reported covering the broad pH range 0.7–12.9, mainly using electron paramagnetic resonance (EPR) methods. It is tested to which extent the pH-induced conformational isomers of HSA reveal themselves in continuous wave (CW) EPR spectra from this spin probing approach in comparison to an established spin-labeling strategy utilizing 3-maleimido proxyl (5-MSL). Most analyses are conducted on empirical levels with robust strategies that allow for the detection of dynamic changes of ligand, as well as protein. Special emphasis has been placed on the EPR spectroscopic detection of a molten globule (MG) state of HSA that is typically found by the fluorescent probe 8-Anilino- naphthalene-1-sulfonic acid (ANS). Moreover, four-pulse double electron-electron resonance (DEER) experiments are conducted and substantiated with dynamic light scattering (DLS) data to determine changes in the solution shape of HSA with pH. All results are ultimately combined in a detailed scheme that describes the pH-induced functional phase space of HSA.

Electron paramagnetic resonance of a copper doped [(CH3)2NH2][Zn(HCOO)3] hybrid perovskite framework.

We report a continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) as well as pulse electron nuclear double resonance (ENDOR) study of Cu2+ doped [(CH3)2NH2][Zn(HCOO)3] hybrid perovskite which exhibits a structural phase transition. The multifrequency (X, Q and W-band) CW EPR measurements allow the temperature evolution of the Cu2+ ion local environment to be studied. The spectrum of the ordered (low-temperature) phase reveals an axially distorted octahedral Cu2+ site confirming the successful replacement of the Zn2+ ions and formation of the CuO6 octahedra. The CW EPR spectrum of the disordered (high-temperature) phase shows an additional broad line which gradually diminishes on cooling. The EPR linewidth of the axially symmetric Cu2+ ion site exhibits an anomaly at the phase transition point and Arrhenius-type behavior in the disordered phase. The temperature dependent Cu2+ spin Hamiltonian parameters change abruptly at the phase transition point indicating a strong first-order character of the transition. The X-band pulse ENDOR spectrum of the ordered phase reveals several protons in the vicinity of the Cu2+ center.

Influence of the Nature of the Ion Pairs on the Segmental Dynamics in Polyelectrolyte Complex Coacervate Phases

A spin label has been covalently attached to the weak polyanion poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the segmental dynamics of the polyanion backbone in polyelectrolyte complex coacervate phases formed with oppositely charged polycations such as poly(diallyldimethylammonium chloride) (PDADMAC), linear poly(ethylenimine) (l-PEI) and poly(allylamine hydrochloride) (PAH) by continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy. The segmental rotational mobility of the spin-labeled polyanion has been determined by the simulation of the line shape of the experimental CW EPR spectra using the microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion. The study has shown that the segmental mobility of the polyanion backbone in the coacervate phases decreases in the sequence PDADMAC > l-PEI > PAH. The nature of the ion pairs formed between the oppositely charged polyelectrolytes has a strong impact on the dynamics of the polyanion.

Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t-test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant (P ≤ 0.05) and can be attributed to sources other than preparation/technique.

Tolyporphin Macrocycles from the Cyanobacterium Tolypothrix nodosa Selectively Bind Copper and Silver and Reverse Multidrug Resistance.

Tolyporphins are glycosylated macrocycles isolated from lipophilic soil extracts of the cyanobacterium, Tolypothrix nodosa, and found to potentiate the cytotoxicity of antitumor drugs like vinblastine and adriamycin. Here we find that, unlike porphyrins, tolyporphins are not able to form complexes with most metal ions. However, they do react strongly with copper(II) and silver(II), forming square-planar metal complexes with an unpaired electron in a dx2-y2 orbital of the metal delocalized onto the ligating tolyporphin nitrogen atoms. Complexes were characterized by visible absorption spectra, mass spectrometry (EI, FAB, ESI, LDI-TOF, and MALDI-TOF) and multifrequency continuous-wave electron paramagnetic resonance spectra. Copper(II) and silver(II) complexes of tolyporphins A and E were found to have the interesting property of reversing multidrug resistance (MDR), with the copper complexes being less toxic than free tolyporphins. Reactive oxygen-free radicals were implicated in both the cytotoxic and MDR-reversing effects of free and metalated tolyporphins.

Simulating Electron Paramagnetic Resonance Spectra of Slow-Motion Systems in the Time Domain

Electron paramagnetic resonance (EPR) spectroscopy has proven to be an excellent tool for probing local structure and dynamics in biological systems. However, the corresponding spectra can be complex and difficult to interpret. In order to accurately interpret experimental data, there has been a corresponding active development of computational tools and user-friendly programs to simulate continuous-wave (CW) EPR spectra, especially those in the slow-motion regime (dynamical time scales of ≈10-100 ns for nitroxides at 9-10 GHz). Though the standard method has been to numerically solve the stochastic Liouville equation in the frequency domain, more recently, simulating CW EPR spectra in the time domain has become increasingly popular. Existing works have applied this method by simulating spin relaxation due to rotational diffusion, either by generating stochastic or molecular dynamics trajectories. Other significant spin relaxation mechanisms that affect CW EPR spectral lineshapes have been treated in a phenomenological manner. In order to bridge this gap in computational complexity and more accurately simulate EPR spectra obtained in experiments, we extend previous treatments by explicitly including other spin relaxation mechanisms and explore additional motional models. In addition, our programs are implemented in the widely used Matlab software environment.

EPR of Structural Phase Transition in Manganese- and Copper-Doped Formate Framework of [NH3(CH2)4NH3][Zn(HCOO)3]2

Electron paramagnetic resonance (EPR) and pulse electron–nuclear double resonance (ENDOR) spectroscopies are applied to investigate the structural phase transition and the low temperature phase in manganese- and copper-doped [NH3(CH2)4NH3][Zn(HCOO)3]2 dense metal–organic framework. Continuous-wave (CW) EPR measurements indicate successful incorporation of Mn2+ and Cu2+ ions at the Zn2+ lattice sites. Pulse ENDOR spectrum reveals at least four different proton species in the vicinity of the Mn2+ center showing excellent agreement with the X-ray diffraction experiments. Temperature-dependent CW EPR spectra demonstrate that Mn2+ and Cu2+ local paramagnetic probes are sensitive to the phase transition in the studied compounds. The analysis of the temperature dependence of the Cu2+ hyperfine coupling parameter reveals a first-order phase transition at Tc = 235 K into an antiferroelectric phase that is close to the tricritical point. The obtained logarithmic divergence of the line width of the Mn2+ EPR spectrum...

Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.