Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line.

INTRODUCTION: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. MATERIALS AND METHODS: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. RESULTS: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. CONCLUSIONS: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

Curcumin delivery by modified biosourced carbon-based nanoparticles.

Aim: To prepare a novel hybrid system for the controlled release and delivery of curcumin (CUR). Methods: A method for the ultrasound-assisted fabrication of protein-modified nanosized graphene oxide-like carbon-based nanoparticles (CBNPs) was developed. After being modified with bovine serum albumin (BSA), CUR was loaded onto the synthesized hybrid (labeled CBNPs@BSA-CUR). The structure and properties of the synthesized nanoparticles were elucidated using transmission electron microscopy (TEM), atomic force microscopy (AFM), ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR) and x-ray photoelectron spectroscopy (XPS) methods. Results: CBNPs@BSA-CUR showed pH sensitivity and were calculated as controlled CUR release behavior. The drug-free system exhibited good biocompatibility and was nontoxic. However, CBNPs@BSA-CUR showed acceptable antiproliferative ability against MCF-7 breast cancer cells. Conclusion: CBNPs@BSA-CUR could be considered a highly promising nontoxic nanocarrier for the delivery of CUR with good biosafety.