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.
A method for evaluating mitochondrial membrane potential in isolated leukocyte suspension with the use of sensitive fl uorescent cation-active dye 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine (JC-1) and spectrophotometry is described. JC-1 monomer rapidly penetrates through mitochondrial membrane of living cell and form aggregations characterized by red fl uorescence (λ = 585 nm). In case of mitochondrial membrane depolarization (early sign of apoptosis), JC-1 is not accumulated in the mitochondria and is present in the cytoplasm as a monomer characterized by green spectral fl uorescence (λ = 510 nm). The method can be used for evaluation of the function of living cells and mechanisms regulating energy metabolism by evaluating the mitochondrial membrane potential.
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