P.F508del editing in cells from cystic fibrosis patients

PLoS One. 2020 Nov 11;15(11):e0242094. doi: 10.1371/journal.pone.0242094. eCollection 2020.

Abstract

Development of genome editing methods created new opportunities for the development of etiology-based therapies of hereditary diseases. Here, we demonstrate that CRISPR/Cas9 can correct p.F508del mutation in the CFTR gene in the CFTE29o- cells and induced pluripotent stem cells (iPSCs) derived from patients with cystic fibrosis (CF). We used several combinations of Cas9, sgRNA and ssODN and measured editing efficiency in the endogenous CFTR gene and in the co-transfected plasmid containing the CFTR locus with the p.F508del mutation. The non-homologous end joining (NHEJ) frequency in the CFTR gene in the CFTE29o- cells varied from 1.25% to 2.54% of alleles. The best homology-directed repair (HDR) frequency in the endogenous CFTR locus was 1.42% of alleles. In iPSCs, the NHEJ frequency in the CFTR gene varied from 5.5% to 12.13% of alleles. The best HDR efficacy was 2.38% of alleles. Our results show that p.F508del mutation editing using CRISPR/Cas9 in CF patient-derived iPSCs is a relatively rare event and subsequent cell selection and cultivation should be carried out.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Cells, Cultured
  • Cystic Fibrosis / genetics*
  • Cystic Fibrosis Transmembrane Conductance Regulator / genetics*
  • DNA Repair
  • Gene Editing / methods*
  • Humans
  • Induced Pluripotent Stem Cells / metabolism

Substances

  • cystic fibrosis transmembrane conductance regulator delta F508
  • Cystic Fibrosis Transmembrane Conductance Regulator

Grants and funding

The sections about iPSCs culture obtaining and editing were supported by the grant of the Russian Science Foundation (Agreement 17-75-20095), the other sections were obtained within the state assignment of Ministry of Science and Higher Education of Russian Federation.