BIOMEDICAL JOURNAL OF PIROGOV RNRMU (MOSCOW, RUSSIA)
Experimental approaches to the target editing of the CFTR gene using CRISPR-Cas9
Cystic fibrosis is a severe autosomal recessive disease caused by mutations in the CFTR gene. The most common CFTR mutation occurring in the European population is F508del. Advances in the management of patients with cystic fibrosis aimed at blocking disease progression have considerably improved the prognosis, but gene therapy has turned to be less effective than expected. Capable of correcting mutations direct in the cells, genome editing, and specifically the CRISPR-Cas9 technology, raises hope of causal treatment for patients with cystic fibrosis. The aim of this work was to compare and improve the efficacy of F508del editing using different combinations of guide RNAs and Cas9. The study was carried out in HEK293T cells. The efficacy of editing was assessed for both plasmid and genomic sites by T7E1 analysis. The best effect was demonstrated by a combination of SaCas9 and sgRNA targeting F508del: 29% of alleles were successfully edited. A combination of SpCas9 and a similar sgRNA showed low efficacy due to the low expression of this guide RNA. All attempts to improve its expression failed. SgRNA stabilization by introducing a G-quadruplex into the sgRNA sequence and adding GG to the 5′-region also did not work. Perhaps, low performance of this guide RNA is determined by its nucleotide sequence, limiting its use.