Fig. 6: Shuttle peptide delivery of ABE8e-Cas9 RNP to primary air liquid interface cultures of human airway epithelial cells (R553X/L671X) targeting R553X locus.

One week following the first application of ABE8e-Cas9 RNP delivery with shuttle peptides, Ussing chamber analysis was conducted, and DNA editing was analyzed by HTS. a Top panel shows the target DNA strands and PAM sites (blue text). Yellow highlight denotes the predicted 4–8 nt ABE editing window (numbered). Mutations highlighted in red text. b Average frequency of desired product and allelic editing efficiencies for R553X nonsense mutation with the indicated shuttle peptides. Percent allelic editing efficiencies calculated by (% base edited-50)/50 *100 and graphically represented in (c) and the values are highlighted inlarge font. Note that the cells were compound heterozygous for the CFTR R553X mutation. Thus, all cells contain “wild type” sequence for one allele and have the R553X mutation on the other allele. This mathematical adjustment provides a meaningful “per cell” estimate of correction. Statistics by ordinary one-way ANOVA without any adjustments, ***P < 0.0001. d, e CFTR-dependent anion channel activity summarized from short circuit current tracings across all treatment groups. Change in short circuit current (ΔIsc) in response to F&I (forskolin & IBMX) (d) and GlyH (e) in groups represented in (c) and non-CF donor cells. Statistics by ordinary one-way ANOVA without any adjustments, *P = 0.01, **P = 0.003. f Representative short circuit current tracings comparing mock, ABE8e-Cas9 RNP alone ABE8e-Cas9 RNP + S10 (blue), and ABE8e-Cas9 RNP + S315 (red) treated cells. For (c–e), the CF human airway epithelia are represented by 3 technical replicates (treatment goups) or 4 (Mock group) technical replicates, and non-CF human airway epithelia are represented by 12 biological replicates. Each data point represents one cell culture. Data are plotted as mean ± SEM. Source data are provided as a Source Data File.