Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions.

CRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 µM of Cas9, equimolar Cas9/gRNA ratio and 2 µM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models.

Advances in Genome Editing and Application to the Generation of Genetically Modified Rat Models.

The rat has been extensively used as a small animal model. Many genetically engineered rat models have emerged in the last two decades, and the advent of gene-specific nucleases has accelerated their generation in recent years. This review covers the techniques and advances used to generate genetically engineered rat lines and their application to the development of rat models more broadly, such as conditional knockouts and reporter gene strains. In addition, genome-editing techniques that remain to be explored in the rat are discussed. The review also focuses more particularly on two areas in which extensive work has been done: human genetic diseases and immune system analysis. Models are thoroughly described in these two areas and highlight the competitive advantages of rat models over available corresponding mouse versions. The objective of this review is to provide a comprehensive description of the advantages and potential of rat models for addressing specific scientific questions and to characterize the best genome-engineering tools for developing new projects.

Simultaneous photocatalytic Cr(VI) reduction and phenol degradation over copper sulphide-reduced graphene oxide nanocomposite under visible light irradiation: Performance and reaction mechanism.

The contamination of water by synthetic organic molecules and trace metals is a growing challenge, in spite of the enormous research efforts being made in the field of water treatment. In this study, reduced graphene oxide-copper sulphide (rGO-CuS) nanocomposites of different rGO/CuS (2/1, 1/1, 1/2) molar ratios were fabricated via a facile one-step hydrothermal method. The nanocomposite materials, named hereafter as 2rGO-CuS, rGO-CuS and rGO-2CuS, were characterized using various analytical techniques, including X-ray diffraction (XRD), Raman spectroscopy, transmission electron microscopy (TEM), energy dispersive X-ray (EDX) spectroscopy, X-ray photoelectron spectroscopy (XPS) and UV-visible spectrophotometry. The photocatalytic performance of the nanocomposites was assessed under visible light irradiation (λ > 420 nm) for the simultaneous photocatalytic reduction of Cr(VI) and phenol degradation. It was found that rGO-2CuS achieved a remarkable enhancement of the photocatalytic activity among the prepared nanocomposites for the degradation of phenol and reduction of Cr(VI). Therefore, the simultaneous photocatalytic phenol degradation and Cr(VI) reduction over rGO-2CuS sample was further investigated. The experimental results revealed that rGO-2CuS catalyst maintained good degradation efficacy of mixed pollutants after 6 runs and dissolved oxygen was found to be essential to promote Cr(VI) reduction and phenol degradation. A detailed photocatalytic activity under visible light irradiation mechanism was proposed based on quenching experiments and fluorescence measurements.

Purification for Carbon Nanotubes Synthesized by Flame Fragments Deposition via Hydrogen Peroxide and Acetone.

Carbon nanotubes (CNTs) are synthesized by the flame fragment deposition (FFD) technique using Iraqi liquefied petroleum gas (LPG) as a source of carbon in a hand-made reactor at a low temperature (160 °C) without using a catalyst. Purification of the multi-walled carbon nanotubes (MWCNTs) is carried out using a two-step process consisting of sonication in 30 wt.% hydrogen peroxide (H2O2) solution at room temperature to remove amorphous impurities adhering to the walls of the CNTs and carbon nanoparticles (CNPs), followed by sonication in an acetone bath to remove the polyaromatic hydrocarbons (PAH) formed during the LPG gas burning. Comprehensive characterizations such as X-ray diffraction (XRD), atomic force microscopy (AFM), thermo-gravimetric analysis (TGA), and transmission electron microscopy (TEM) were conducted to verify the efficiency of the purification process. The results clearly demonstrated that this process is promising for the purification of the synthesized CNTs.

Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

PMS activation using reduced graphene oxide under sonication: Efficient metal-free catalytic system for the degradation of rhodamine B, bisphenol A, and tetracycline.

This study addresses the influence of ultrasound irradiation on the activation of peroxymonosulfate (PMS) using reduced graphene oxide (rGO) under metal-free conditions for the catalytic degradation of rhodamine B (RhB), bisphenol A (BPA) and tetracycline (TC). Our results revealed that the combination of PMS/rGO and ultrasonication enhanced significantly the degradation rate, reaching full degradation in relatively short times with total organic carbon (TOC) removal exceeding 85% of the investigated pollutants. In contrast, under these experimental conditions, rGO/ultrasound and PMS/ultrasound achieved less than 20% degradation of the same pollutants. Electron paramagnetic resonance (EPR) studies along with quenching experiments suggested that hydroxyl radicals (OH) are the dominant reactive species in the degradation process. Furthermore, inductively coupled plasma atomic emission spectroscopy (ICP-AES) and EPR data revealed the presence of trace manganese (Mn) in rGO. To elucidate the role of Mn on the degradation process, rGO was subjected to hot acid treatment for 48 h to remove trace Mn. While the chemical composition of rGO was not significantly altered by this chemical treatment, the degradation efficiency decreased upon Mn dissolution. The result suggests that trace metal in rGO might account for the efficiency of PMS activation. Finally, plausible degradation pathways were proposed based on LC-MS analysis of the reaction intermediates.

CRISP-ID: decoding CRISPR mediated indels by Sanger sequencing.

The advent of next generation gene editing technologies has revolutionized the fields of genome engineering in allowing the generation of gene knockout models and functional gene analysis. However, the screening of resultant clones remains challenging due to the simultaneous presence of different indels. Here, we present CRISP-ID, a web application which uses a unique algorithm for genotyping up to three alleles from a single Sanger sequencing trace, providing a robust and readily accessible platform to directly identify indels and significantly speed up the characterization of clones.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins.

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

Procedures for somatic cell nuclear transfer in the rat.

Somatic cell nuclear transfer (SCNT) is a powerful tool for the investigation of the mechanisms of nuclear remodeling. In addition, SCNT may offer the possibility of introducing targeted mutations by homologous recombination in species for which ES cell technology is not available. The rat specific features of the oocyte have long impeded the development of SCNT. We detail here the procedures developed and optimized during the last several years for the optimization of rat cloning.

Pronuclear DNA injection for the production of transgenic rats.

In the absence of germ-line-competent ES cells in the rat, pronuclear microinjection remains an essential tool to generate transgenic rat models. However, DNA microinjection procedures first developed for mouse do not provide scientists with satisfying results when applied to rat. Here we describe optimized procedures for rat with a special focus on rat embryo production, in vitro incubation and culture, DNA pronuclear injection, and the transfer of embryos into foster females.