DOI: 10.24075/brsmu.2018.022


Experimental approaches to the target editing of the CFTR gene using CRISPR-Cas9

Smirnikhina SA1, Anuchina AA1, Kochergin-Nikitsky KS1, Adilgereeva EP1, Yakushina VD1, Lavrov AV1,2
About authors

1 Laboratory of Mutagenesis,
Research Centre for Medical Genetics, Moscow

2 Department of Molecular and Cellular Genetics, Biomedical Faculty,
Pirogov Russian National Research Medical University, Moscow

Correspondence should be addressed: Svetlana Smirnikhina
Moskvorechie 1, Moscow, 115522; moc.liamg@sanihkinrims

About paper

Funding: the section Editing of the CFTR locus was supported by the grant of the Russian Science Foundation (Agreement 17-75-20095), the sections Increasing the expression of guide RNAs and Improving the efficacy of CFTR locus editing were supported by the Russian Academy of Sciences and the state assignment of FASO Russia.

Received: 2018-03-15 Accepted: 2018-03-20

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.

Keywords: genome editing, cystic fibrosis, CFTR, F508del mutation, guide RNA, CRISPR-Cas9