Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells

Abstract

Prime editing is a recent, CRISPR-derived genome editing technology capable of introducing precise nucleotide substitutions, insertions, and deletions. Here, we present prime editing approaches to correct L227R- and N1303K-CFTR, two mutations that cause cystic fibrosis and are not eligible for current market-approved modulator therapies. We show that, upon DNA correction of the CFTR gene, the complex glycosylation, localization, and, most importantly, function of the CFTR protein are restored in HEK293T and 16HBE cell lines. These findings were subsequently validated in patient-derived rectal organoids and human nasal epithelial cells. Through analysis of predicted and experimentally identified candidate off-target sites in primary stem cells, we confirm previous reports on the high prime editor (PE) specificity and its potential for a curative CF gene editing therapy. To facilitate future screening of genetic strategies in a translational CF model, a machine learning algorithm was developed for dynamic quantification of CFTR function in organoids (DETECTOR: "detection of targeted editing of CFTR in organoids").

Keywords: CRISPR; DETECTOR; cystic fibrosis; gene editing; human nasal epithelial cells; machine learning; patient-derived organoids; prime editing.

Graphical abstract

Figure 1

Prime editing of c.680T>G (L227R) rescues CFTR glycosylation, PM localization, and anion channel function (A) PE3 strategies require delivery of the prime editor (PE) enzyme, prime editing guide (pegRNA), and nicking guide RNA (ngRNA). pegRNAs consist of a spacer, single guide RNA (sgRNA) scaffold, reverse-transcriptase template (RTT), and primer binding site (PBS) sequences. (B) L227R locus with pegRNA+13C>A designs designed to reverse c.680T>G. (C) DNA correction in 3HA-L227R-CFTR HEK293T cells following plasmid transfection of different pegRNAs and prime editing strategies (PE2 vs. PE3). Tukey’s multiple comparisons test did not reveal significant differences between the best-performing +3G>T and +13C>A pegRNAs. (D) Percentage of cells with PM-localized CFTR correlates with CFTR correction on DNA level. Spearman correlation r = 0.8182, p < 0.0001. PM staining and flow cytometry using antibodies against the extracellular 3HA tag of 3HA-L227R-CFTR HEK293T cells. (E) Percentage of 3HA-L227R-CFTR HEK293T cells positive for PM-CFTR following plasmid transfection of different pegRNAs and prime editing strategies (PE2 vs. PE3). (F) Western blot of CFTR-negative (NT), control- or L227R PE3-treated 3HA-L227R-CFTR, or wild-type (WT)-CFTR HEK293T cell lines. Bands B and C, respectively, represent core- and complex-glycosylated CFTR. (G) Immunocytochemistry staining and confocal microscopy of HEK293T NT, control or L227R-targeted 3HA-L227R-CFTR, and 3HA-WT-CFTR cells. DAPI-stained nuclei are represented in blue, and labeling of extracellular 3HA-CFTR is shown in green. (H) Halide-sensitive yellow fluorescent protein (HS-YFP) quenching assay in NT or L227R- or WT-CFTR HEK293T cells treated with L227R-targeting or control PE3 approaches. Traces and error bars represent n = 3 biological repeats. (I) 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 = non-CFTR-targeting pegRNA + ngRNA (RNF2 locus). Tukey’s multiple comparisons test was used to compare experimental conditions. All data are presented as mean ± S.D. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, or ∗∗∗∗p < 0.0001.

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-CFTR^high+ 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.

Figure 3

Prime editing of the endogenous CFTR locus restores CFTR function in patient-derived rectal organoids (A) Forskolin-induced swelling (FIS) in rectal organoids is a CFTR-dependent/-specific phenotype and hence well suited to evaluate functional correction after prime editing. (B) Representative images from FIS experiments in organoids homozygous for L227R or N1303K transduced with lentiviral vectors (LVs) encoding PE enzyme, pegRNA, and ngRNA. (C) To assess individual organoid swelling, a machine learning-based algorithm was developed: DETECTOR (detection of targeted editing of CFTR in organoids). The tool first counts all organoids and subsequently evaluates the number of swelling organoids based on an image that overlays individual organoid areas at the start and end of the FIS assay (difference image). (D) Percentage of FIS-responsive organoids (determined by DETECTOR) in samples from patients homozygous or heterozygous for L227R treated by LV-delivered PE3 approaches. Unpaired parametrical t test with Welch’s correction was used to compare groups. (E) Percentage of FIS-responsive organoids (determined by DETECTOR) in samples from patients homozygous or heterozygous for N1303K. Mann-Whitney U t test was used to compare groups. (F) Percentage of FIS-responsive organoids following co-transduction of LVs containing PE, pegRNA, and ngRNA with an LV expressing MLH1dn (PE5 strategy) in organoids homozygous or heterozygous for N1303K. Unpaired parametrical t test was used to compare groups. Bars represent the mean of two biological replicates measured as technical octuplicates. Control PE3 = delivery of non-CFTR-targeting PE3 approach (RNF2 locus). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, or ∗∗∗∗p < 0.0001.

Figure 4

Prime editing of the endogenous CFTR locus restores CFTR function in 16HBE and primary nasal epithelial cells (A) DNA correction and correction of CFTR function in engineered 16HBEgeN1303K-YFP and 16HBEgeN1303K_MLH1dn-YFP cells transduced with LVs encoding PE enzyme, engineered pegRNA (epegRNA), and ngRNA. CFTR function was determined by HS-YFP quenching and calculated as 1-F/F₀ (with F₀ = fluorescence at point of iodide buffer injection) relative to WT 16HBE-YFP cells. (B and C) Representative short-circuit currents (Isc) of human nasal epithelial (HNE) cells with L227R/L227R (B) or N1303K/R1162X (C) genotypes. HNEs were transduced with LVs containing PE3 or PE5 components and subsequently differentiated at the air-liquid interface (ALI). Arrows indicate the addition of 100 μM 3-isobutyl-1-methylxanthine (IBMX) + 10 μM forskolin (Fsk) or 10 μM Inh172. (D and E) Quantified CFTR-dependent Isc (ΔFsk/IBMX and ΔInh172) for PE3 or PE5 of (D) L227R homozygous and WT HNEs and (E) N1303K/N1303K, N1303K/G1182X, and WT HNEs. Bars represent n = 5 or n = 3 biological repeats, and error bars represent SD. WT HNEs are the same in (D) and (E). Tukey’s multiple comparisons test was used to compare experimental conditions. Control PE3/PE5 = delivery of non-CFTR-targeting pegRNA + ngRNA (RNF2 locus).

Figure 5

Off-target profiling of L227R and N1303K prime-editing guides (A and B) Potential SpCas9 off-target sites identified through GUIDE-seq in HEK293T cells using SpCas9 combined with (A) L227R and (B) N1303K pegRNAs and ngRNAs. (C and D) Targeted deep sequencing on potential off-target sites identified by in silico prediction and/or GUIDE-seq. HEK293T samples were collected 3 days after transfection. Organoid samples were harvested 14 days post-transduction. Bars represent mean percentage of modified reads evaluated at expected nick site ± SD of two biological replicates. Control PE3/PE5 = delivery of non-CFTR-targeting pegRNA + ngRNA (RNF2 locus).