Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

There have been many engineered Cas9 variants that were developed to minimize unintended cleavage of off-target DNAs, but detailed mechanism for the way they regulate the target specificity through DNA:RNA heteroduplexation remains poorly understood. We used single-molecule FRET assay to follow the dynamics of DNA:RNA heteroduplexation for various engineered Cas9 variants with respect to on-target and off-target DNAs. Just like wild-type Cas9, these engineered Cas9 variants exhibit a strong correlation between their conformational structure and nuclease activity. Compared with wild-type Cas9, the fraction of the cleavage-competent state dropped more rapidly with increasing base-pair mismatch, which gives rise to their enhanced target specificity. We proposed a reaction model to quantitatively analyze the degree of off-target discrimination during the successive process of R-loop expansion. We found that the critical specificity enhancement step is activated during DNA:RNA heteroduplexation for evoCas9 and HypaCas9, while it occurs in the post-heteroduplexation stage for Cas9-HF1, eCas9, and Sniper-Cas9. This study sheds new light on the conformational dynamics behind the target specificity of Cas9, which will help strengthen its rational designing principles in the future.

Decoration of CuO NWs Gas Sensor with ZnO NPs for Improving NO2 Sensing Characteristics.

This paper introduces a method for improving the sensitivity to NO2 gas of a p-type metal oxide semiconductor gas sensor. The gas sensor was fabricated using CuO nanowires (NWs) grown through thermal oxidation and decorated with ZnO nanoparticles (NPs) using a sol-gel method. The CuO gas sensor with a ZnO heterojunction exhibited better sensitivity to NO2 gas than the pristine CuO gas sensor. The heterojunction in CuO/ZnO gas sensors caused a decrease in the width of the hole accumulation layer (HAL) and an increase in the initial resistance. The possibility to influence the width of the HAL helped improve the NO2 sensing characteristics of the gas sensor. The growth morphology, atomic composition, and crystal structure of the gas sensors were analyzed using field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy, and X-ray diffraction, respectively.

Comparative Single-Molecule Kinetic Study for the Effect of Base Methylation on a Model DNA-Protein Interaction.

We studied how the interaction between HindIII endonuclease and dsDNA is affected by the single-base modification of the latter by a single-molecule kinetic assay. For a comparative study of chemical modifications, we measured the binding and unbinding rates of the HindIII-DNA complex for normal dsDNA, methylated DNA, and hydroxymethylated DNA. We found that methylation of DNA at the recognition site results in a large increase in the unbinding rate due to the steric effect, which is consistent with the standard free energy change in the transition state. On the contrary, methylation minimally affects the binding rate, as simultaneous increases in the activation energy and the pre-exponential factor compensate for each other.

Sensitivity Improvement of Urchin-Like ZnO Nanostructures Using Two-Dimensional Electron Gas in MgZnO/ZnO.

This paper introduces a strategy for improving the sensitivity of a gas sensor to NO2 gas. The gas sensor was fabricated using urchin-like ZnO nanostructures grown on MgO particles via vapor-phase growth and decorated with MgZnO nanoparticles via a sol-gel process. The urchin-like ZnO gas sensor decorated with MgZnO showed higher sensitivity to NO2 gas than a pristine urchin-like ZnO gas sensor. When ZnO and MgZnO form a heterojunction, a two-dimensional electron gas is generated. This improves the performance of the fabricated gas sensor. The growth morphology, atomic composition, and phase structure were confirmed through field-emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray diffraction, respectively.

Positive Identification of DNA Cleavage by CRISPR-Cas9 Using Pyrene Excimer Fluorescence to Detect a Subnanometer Structural Change.

The Cas9 nuclease binds and cleaves DNA through its large-scale structural rearrangements. However, its unique property of not releasing the cleaved DNA has forbidden spectroscopic detection of the cleavage event. Here, we employ a novel fluorescence probe based on pyrene excimer emission to detect a minute structural change not detectable by other methods and demonstrate its applicability to spectroscopic tracking of the Cas9 nuclease activity in time. We show that the intensity of excimer emission depends sensitively on a subtle change in the structural environment of the target nucleic acid, which enables discrimination between cleaved and uncleaved nucleic acids within the DNA/Cas9/gRNA ternary complex. Kinetic parameters were obtained from the temporal evolution of the excimer emission, which revealed that DNA binding is hardly affected by PAM-distal mismatches, whereas the rate of cleavage by Cas9 decreases dramatically even with a 1-bp mismatch. Spectroscopic studies using the pyrene-based probe should be promising for biomolecular systems affected by subnm structural changes.

Improved Sensitivity of α-Fe2O3 Nanoparticle-Decorated ZnO Nanowire Gas Sensor for CO.

A strategy for improving the sensitivity of a sensor for detecting CO and NH3 gases is presented herein. The gas sensor was fabricated from ZnO metal oxide semiconductor nanostructures grown via a vapor⁻liquid⁻solid process and decorated with α-Fe2O3 nanoparticles via a sol⁻gel process. The response was enhanced by the formation of an α-Fe2O3/ZnO n⁻n heterojunction and the growth of thinner wires. ZnO nanowires were grown on indium⁻tin⁻oxide glass electrodes using Sn as a catalyst for growth instead of Au. The structure and elemental composition were investigated using field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction. The gas sensing results indicate that the response value to 100 ppm CO was 18.8 at the optimum operating temperature of 300 °C.

General and facile purification of dye-labeled oligonucleotides by pH-controlled extraction.

Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. When the pH of an aqueous solution containing dye-labeled DNA and unreacted free dye is lowered, and the solution is mixed with a hydrophobic organic solvent (butanol), the neutral free dye is preferentially dissolved in the organic phase, leaving behind the hydrophilic dye-labeled DNA in the aqueous phase. We experimentally verified that our new method results in high yields of dye-labeled oligonucleotides and the efficient removal of free dye.

High neutrophil : lymphocyte ratio is associated with refractory Kawasaki disease.

BACKGROUND: The clinical significance of the neutrophil : lymphocyte ratio (NLR) has not yet been fully elucidated in Kawasaki disease (KD). The purpose of this study was to investigate the relationship between NLR and response to i.v. immunoglobulin (IVIG), and its effect on coronary abnormalities in KD. METHODS: A total of 196 KD patients treated with IVIG were analyzed. Baseline NLR was evaluated immediately before IVIG therapy and the patients classified into two groups according to NLR. The clinical data, other inflammatory biomarkers, and coronary complications were also assessed. RESULTS: Kawasaki disease patients with NLR ≥ 5 had a greater incidence of IVIG refractoriness than the NLR < 5 group (31.7% vs 4.3%, P < 0.001), but this was not related to the development of coronary abnormalities. The change in NLR after IVIG (i.e. ΔNLR) was significantly decreased in the coronary abnormality group (2.65 ± 1.88 vs 3.81 ± 2.55, P = 0.042). On multivariate analysis, high NLR and CRP were independent predictors of IVIG refractoriness during the acute phase of KD (P = 0.032 in NLR; P = 0.029 in CRP, respectively). CONCLUSIONS: High NLR was closely associated with resistance to IVIG, but it was not related to the occurrence of coronary abnormalities in KD. Low ΔNLR after IVIG, however, was significantly associated with coronary artery abnormalities.

Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease.

The type II CRISPR-associated protein Cas9 recognizes and cleaves target DNA with the help of two guide RNAs (gRNAs; tracrRNA and crRNA). However, the detailed mechanisms and kinetics of these gRNAs in the Cas9 nuclease activity are unclear. Here, we investigate the structural roles of gRNAs in the CRISPR-Cas9 system by single-molecule spectroscopy and reveal a new conformation of inactive Cas9 that is thermodynamically more preferable than active apo-Cas9. We find that tracrRNA prevents Cas9 from changing into the inactive form and leads to the Cas9:gRNA complex. For the Cas9:gRNA complex, we identify sub-conformations of the RNA-DNA heteroduplex during R-loop expansion. Our single-molecule study indicates that the kinetics of the sub-conformations is controlled by the complementarity between crRNA and target DNA. We conclude that both tracrRNA and crRNA regulate the conformations and kinetics of the Cas9 complex, which are crucial in the DNA cleavage activity of the CRISPR-Cas9 system.