CORRECTION OF THE MUTATION CAUSE OF CYSTIC FIBROSIS (F508DEL) IN PULMONARY EPITHELIUS LINE CELLS BY CRISPR/CAS9

Abstract

Cystic fibrosis is a genetic, autosomal recessive disease caused by mutations in the CFTR gene (cystic fibrosis transmembrane conductance regulator). The most common mutation is F508del with around 70% prevalence worldwide. Currently, the disease has no cure and the main symptoms are treated. The technique of the CRISPR/Cas has favorable prospects to correct the mutation in DNA and rescue function of CFTR protein, improving patient's quality of life. The main objective this work was to build molecular tools to correct the F508del mutation of cystic fibrosis in a pulmonary epithelial cell line (CFBE). The work is divided into stages: (1) Design of sgRNA and construction of tools and (2) edition evaluation by qPCR, PCR and Sanger sequencing techniques. The designs sgRNAs were selected based on the proximity of the mutation, number of off-targets and efficiency predicted. The insertion of the sgRNA sequence in the plasmid (px458) was confirmed by the PCR technique, digestion with restriction enzymes and sequencing. The transfection method selected was by lipid agents, showing an efficiency of about 9% in the entry of the plasmid into target cell. The built tool based ribonucleoprotein, had the RNA obtained by in vitro transcription. After formation of the complex with the purified protein (saCas9) this tool showed activity (30%) in recognizing and cleaving DNA in vitro. After 48 hours transfection cells, was made a sorting by flow cytometry and the correction confirmed by qPCR techniques, the detection the correction of the F508del mutation was below 12.5% or null, under the conditions tested. New assays will be performed included design modifications for this tool to obtain satisfactory data.