Correction of F508del in CFTR using CRISPR/Cas9 in cells derived from cystic fibrosis patients

Abstract

BACKGROUND: Cystic fibrosis (CF) is the most common fatal and incurable genetic disease. The most frequent reason is the F508del mutation in the CFTR gene, which can theoretically be corrected by genome editing. Currently, pressing issue is the search for the most effective CRISPR/Cas9-based editing system for this mutation, as well as the selection of target cells that could support self-renewal and provide differentiated progeny of lung cells. Such promising targets can be presented by human induced pluripotent stem cells (hiPSCs) and human induced airway basal stem cells (hiBCs).

AIM: To correct the F508del mutation in the CFTR gene in hiPSCs and hiBCs derived from CF patients using CRISPR/Cas9 technology.

MATERIALS AND METHODS: We obtained three hiPSCs lines by reprogramming fibroblasts from patients with CF and three hiBCs lines by targeted differentiation of these hiPSCs. Based on three different variants of the Cas9 nuclease, three single guide RNAs, and two single-stranded oligodeoxyribonucleotides we created eight systems for editing the F508del mutation and transfected them into hiPSCs and hiBCs by electroporation. Then we analyzed the levels of non-homologous end joining, indels, and directed homologous repair in the transfected cells by deep target sequencing.

RESULTS: Eight editing systems were tested on hiPSCs. The highest efficiency of non-homologous end joining, indels, and directed homologous repair was observed when using SpCas9(1.1). The mutation was significantly corrected using a combination of this nuclease with the guide RNA sgCFTR#sp1 (with an efficiency up to 6.6% of alleles in transfected cells). Only two editing systems), which seemed to be most effective on chiPSCs, were tested on hiBCs. The mutation was significantly corrected using both systems (with efficiency up to 20% of alleles in transfected cells).

CONCLUSION: We demonstrated the possibility of highly effective correction of the F508del mutation in the CFTR gene in cells obtained from patients with CF using a designed system for editing this mutation based on the CRISPR/Cas9 system. Since hiBCs can be transfected and edited quite successfully, and can also be obtained in satisfactory quantities from hiPSCs, they are a promising platform for the development of gene therapy for cystic fibrosis.

Keywords

cystic fibrosis / induced pluripotent stem cells / CRISPR/Cas9 / gene editing / F508del

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