P.F508del editing in cells from cystic fibrosis patients

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.

Fig 1. sgRNAs and ssODNs for the CFTR locus.

The numbers in the nucleotide sequence indicate the number of nucleotides up- and downstream of the target locus. The dashed lines indicate the locations of the expected Cas9-induced DNA double strand breaks. PAMs are marked in blue (note that for the most 5’ PAM its complementary strand is marked); the nucleotides correcting the p.F508del mutation are marked in red; the silent variant in sp_ssODN#2 is marked in green. sgRNA–single guide RNA, ssODN–single-stranded oligodeoxynucleotide.

Fig 2. CFTR gene editing in CFTE29o- cells.

Cells were transfected with various combinations of Cas9, sgRNA and ssODN. DNA (n = 2) was isolated 48–72 hours after transfection and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM.

Fig 3. CFTR gene editing in iPSCs.

Cells were transfected with different combinations of Cas9, sgRNA and ssODN; DNA (n = 2 or n = 1) was isolated after 48–72 hours and the CFTR fragment was amplified from the genomic and plasmid loci; a sequencing library was prepared and then sequenced using a MiSeq System (Illumina); the results were analyzed using the CRISPResso2 software. The Kruskal-Wallis test was used to analyze differences. A. NHEJ in the plasmid and genomic loci in the CFTR gene. B. HDR (p.F508del correction) in the plasmid and genomic loci in the CFTR gene. Results represented as mean ± SEM. iPSCs–induced pluripotent stem cells.