Search

The potential for off-target mutations is a critical concern for the therapeutic application of CRISPR-Cas9 gene editing. Current detection methodologies, such as GUIDE-seq, exhibit limitations in oligonucleotide integration efficiency and sensitivity, which could hinder their utility in clinical settings. To address these issues, we introduce OliTag-seq, an in-cellulo assay specifically engineered to enhance the detection of off-target events. OliTag-seq employs a stable oligonucleotide for precise break tagging and an innovative triple-priming amplification strategy, significantly improving the scope and accuracy of off-target site identification. This method surpasses traditional assays by providing comprehensive coverage across various sgRNAs and genomic targets. Our research particularly highlights the superior sensitivity of induced pluripotent stem cells (iPSCs) in detecting off-target mutations, advocating for using patient-derived iPSCs for refined off-target analysis in therapeutic gene editing. Furthermore, we provide evidence that prolonged Cas9 expression and transient HDAC inhibitor treatments enhance the assay's ability to uncover off-target events. OliTag-seq merges the high sensitivity typical of in vitro assays with the practical application of cellular contexts. This approach significantly improves the safety and efficacy profiles of CRISPR-Cas9 interventions in research and clinical environments, positioning it as an essential tool for the precise assessment and refinement of genome editing applications. OliTag-seq, a specific and reproducible in-cellulo assay for CRISPR/Cas9 off-target analysis, can improve site cleavage efficiency and the identification of off-target sites. [ABSTRACT FROM AUTHOR]

The sol-gel transition of methylcellulose (MC) solutions in the presence of ortho-methoxycinnamic acid (OMCA) or cetyltrimethylammonium bromide (CTAB) and in the coexistence of OMCA and CTAB was determined by the rheological measurement. It has been found that the sol-gel transition temperature of MC solutions increases linearly with the concentration of either OMCA or CTAB in solution, respectively. However, in the coexistence of OMCA and CTAB, the sol-gel transition temperature of MC solutions remains invariable, independent of the concentration of CTAB in solution. The experimental results show that OMCA has priority to adsorb on the methyl group of MC chains to form polymer-bound aggregates. In particular, these aggregates inhibit the hydrophobic interaction between CTAB and the methyl group of MC chains completely. Taking into account the fact that OMCA is almost insoluble in MC-free solutions but dissolves very well in aqueous MC solutions, we propose the formation of the core-shell architecture prompted by OMCA and the methyl group of MC chains, with the methyl group of MC chains serving as the core and the self-assembly of OMCA molecules serving as the shell. Obviously, the formation of the core-shell structure increases the solubility of OMCA, improves the stability of methyl groups of MC chains at high temperatures and inhibits the hydrophobic interaction between CTAB and the methyl group of MC chains in solution. The abnormal behavior relating to the sol-gel transition of MC solutions in the presence of OMCA or in the coexistence of OMCA and CTAB is therefore explained. Upon UV irradiation, the sol-gel transition temperature of MC solutions in the presence of OMCA, or in the coexistence of OMCA and CTAB, decreases notably. However, the dependence of the sol-gel transition temperature of MC solutions as a function of OMCA concentration, or CTAB concentration in the presence of OMCA, does not change after UV irradiation. [ABSTRACT FROM AUTHOR]

CuO/Ce0.8Zr0.2O2 and CuO/CeO2 catalysts were prepared via a impregnation method characterized by using FT-Raman, XRD, XPS and H2-TPR technologies. The catalytic activity of the samples for low-temperature CO oxidation was investigated by means of a microreactor-GC system. The influence of the calcination temperature and different supports on the catalytic activity was studied. [ABSTRACT FROM AUTHOR]

Two compounds, [Co(bpy)2(NiL)](ClO4)2(1)and {[Co(phen)3](NiL)}(ClO4)2(2), have been synthesized: (H2L=2,3-dioxo-5,6,14,15-dibenzo-1,4,8,12-tetraazacylotetradeca-7,12-diene, bpy=2,2'-bypyridine and phen = 1,10-phenanthroline). Compound (1) contains a discrete binuclear [Co(bpy)2(NiL)]2+ cation, while compound (2) is composed of [Co(phen)3]2+ cations and NiL neutral fragments. The magnetic properties of compound (1) have been dealt with by isotropic one-ion approximation of the CoII ion with a spin--orbit coupling in an Oh symmetry environment. The electronic spectra of these two compounds have also been discussed. [ABSTRACT FROM AUTHOR]

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5'-upstream sequence of the rice metallothionein gene ( ricMT) fused to the ß-glucuronidase ( GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm. [ABSTRACT FROM AUTHOR]