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.

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.

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.

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 (HO) 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.

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.

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.

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.

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.

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.

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.