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. 2024 May 1;5(5):101544. doi: 10.1016/j.xcrm.2024.101544

Figure 2.

Figure 2

Prime editing of c.3909C>G (N1303K) rescues CFTR glycosylation, PM localization, and channel function

(A) DNA correction in 3HA-N1303K-CFTR HEK293T cells following plasmid transfection of different pegRNAs and prime-editing strategies (PE2 vs. PE3).

(B) Western blot of CFTR-negative (NT), 3HA-N1303K-CFTR, or WT-CFTR HEK293T cell lines treated with control PE3, N1303K PE3, elexa-teza-ivacaftor (ETI; 3 μM of each, 24 h incubation), or DMSO control. Bands B and C respectively represent core- and complex-glycosylated CFTR.

(C) Immunocytochemistry staining and confocal microscopy of HEK293T NT, 3HA-WT-CFTR, and control- or N1303K-PE3-treated 3HA-N1303K-CFTR cells. DAPI-stained nuclei are represented in blue, and labeling of extracellular 3HA-CFTR is shown in green.

(D) Percentage of PM-CFTRhigh+ PE3-treated 3HA-N1303K-CFTR cells relative to WT. High+ cells were gated based on non-treated 3HA-N1303K HEK293T cells. Gating strategy is shown in Figure S2H.

(E) HS-YFP quenching assay of control- or N1303K PE3-treated HEK293T expressing no CFTR (NT), N1303K-CFTR, or WT-CFTR. CFTR function is calculated as 1-F/F0 (with F0 = fluorescence at point of iodide buffer injection) with subtraction of quenching in NT cells (aspecific/background) and relative to WT-CFTR HEK293T cells. Control PE3 = delivery of non-CFTR-targeting pegRNA + ngRNA (RNF2 locus). All data points reflect biological replicates, presented as mean ± SD. Tukey’s multiple comparisons test was used to compare experimental conditions. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, or ∗∗∗∗p < 0.0001.