Genetic modification of cystic fibrosis with ΔF508 mutation of CFTR gene using the CRISPR system in peripheral blood mononuclear cells.

OBJECTIVES: Cystic fibrosis (CF) is an inherited autosomal recessive disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The present study aimed to investigate the genetic modification of CF with ΔF508 mutation of the CFTR gene using CRISPR in peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Two single guide RNAs were designed to target sequences in the CFTR gene. The transfection efficiency of PBMC cells was examined through evaluation of green fluorescent protein (GFP) expression using fluorescent microscopy. Moreover, a sgRNA-Cas9 plasmid was tested to target the CFTR gene. The ΔF508 gene modification was evaluated and confirmed by PCR and Sanger sequencing methods. RESULTS: Our results indicate the feasibility of site-specific gene targeting with the CRISPR/Cas9 system. 33% of the samples were corrected using CRISPR in mutant locus and confirmed by sequence blast at NCBI databases and primers outside the arm locus. CRISPR/Cas9 approach represents an efficient tool to repair the ΔF508 mutation of the CFTR gene in PBMC Cells. CONCLUSION: Therefore, the CRISPR system can be highly efficient and specific and provides a powerful approach for genetic engineering of cells and model animals. Generally, the proposed method opens new insights into the treatment of human diseases.

CRISPR Genome Editing Technology and its Application in Genetic Diseases: A Review.

Gene therapy has been a long lasting goal for scientists, and there are many optimal methods and tools to correct disease-causing mutations in humans. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been progressively adopted for the assessment a treatment of human diseases, including thalassemia, Parkinson's disease, cystic fibrosis, glaucoma, Huntington's disease, and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS). CRISPR sequences belong to the bacterial immune system, which includes the nuclease Cas enzyme and an RNA sequence. The RNA sequence is unique and pathogen-specific, and identifies and binds to the DNA of invasive viruses, allowing the nuclease Cas enzyme to cut the identified DNA and destroy the invasive viruses. This feature provides the possibility to edit mutations in the DNA sequence of live cells by replacing a specific targeted RNA sequence with the RNA sequence in the CRISPR system. Previous studies have reported the improvement steps in confrontation with human diseases caused by single-nucleotide mutations using this system. In this review, we first introduce CRISPR and its functions and then elaborate on the use of CRISPR in the treatment of human diseases.

Curcumin as a MicroRNA Regulator in Cancer: A Review.

Curcumin is a natural dietary polyphenol for which anti-tumor effects have been documented. Anti-inflammatory and antioxidant properties of curcumin, along with its immunomodulatory, proapoptotic, and antiangiogenic properties, are often referred to as the main mechanisms underlying the anti-tumor effects. At the molecular level, inhibition of NF-kB, Akt/PI3K, and MAPK pathways and enhancement of p53 are among the most important anticancer alterations induced by curcumin. Recent evidence has suggested that epigenetic alterations are also involved in the anti-tumor properties of curcumin. Among these curcumin-induced epigenetic alterations is modulation of the expression of several oncogenic and tumor suppressor microRNAs (miRNAs). Suppression of oncomiRs such as miR-21, miR-17-5p, miR-20a, and miR-27a and over-expression of miR-34 a/c and epithelial-mesenchymal transition-suppressor miRNAs are among the most important effects of curcumin on miRNA homeostasis. The present review will summarize the findings of in vitro and experimental studies on the impact of curcumin and its analogues on the expression of miRNAs involved in different stages of tumor initiation, growth, metastasis, and chemo-resistance.