Fig. 4
From: Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing
Correction of 3849+10kbC>T splicing defect by AsCas12a-crRNA+14 editing. a Scheme of CFTR wild type (pMG3849+10kbWT) and 3849+10KbC>T (pMG3849+10kbC>T) minigenes carrying exon 22, portions of intron 22 encompassing the 3849+10KbC>T, and exon 23 of the CFTR gene. Exons are shown as boxes and introns as lines; the expected spliced transcripts are represented on the right according to the presence or absence of the 3849+10kbC>T mutation. The lower panel shows nucleotide sequence nearby 3849+10kbC>T mutation (labeled in bold) and the AsCas12a-crRNA+14 target position (underlined, with the PAM in red). b Splicing pattern analyzed by RT-PCR in HEK293/pMG3849+10kbC>T cells following treatments with AsCas12a-crRNA control (Ctr) or specific for the 3272-26A>G mutation (+14). Black-solid arrow indicates aberrant splicing, white-empty arrow indicates correct splicing and red triangle indicates a minigene splicing artifact. c Caco-2 cells lentivirally transduced with AsCas12a-crRNA+14 or +14/wt were analyzed for editing in CFTR intron 22 by SYNTHEGO ICE analysis. Data are means ± SEM from n = 2 independent experiments. d Splicing pattern analyzed by RT-PCR in 3849+10kbC>T primary airway cells following lentiviral transduction (15 days) of AsCas12a-crRNA control (Ctr) or specific for the 3849+10kbC>T mutation (+14). Puromycin selection was performed for 72 h in +14 puro. The percentage of aberrant splicing (84 nt insertion into mRNA) was measured by densitometric analysis. e Percentages of indels in 3849+10kbC>T primary airway cells measured by TIDE analysis following lentiviral transduction as in d. Data are from n = 2 independent experiments. f 3849+10Kb C>T patient derived intestinal organoids were lentivirally transduced with AsCas12a-crRNA control (Ctr) or crRNA+14 and analyzed for intron 22 editing by SYNTHEGO ICE analysis. Data are from n = 1 experiment. g Percentage of deep sequencing reads of the edited and non-edited 3849+10kbC>T or WT alleles from f. h Confocal images of calcein green labeled 3849+10KbC>T organoids transduced with AsCas12a-crRNA+14 or CFTR cDNA. Scale bar 200 µm. i Quantification of organoid area; each dot represents the average area of organoids analyzed in each well (number of organoids per well: 3–30) from n = 1 experiment. Data are means ± SD. Statistical analysis was performed using one-way ANOVA; **P < 0.01, n.s. non-significant
