Skip to main content
NCBI home page
PLOS One logoLink to PLOS One
. 2023 Nov 29;18(11):e0295009. doi: 10.1371/journal.pone.0295009

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

Chao Li 1,#, Zhong Liu 1,#, Justin Anderson 2,3, Zhongyu Liu 3, Liping Tang 3,4, Yao Li 3,4, Ning Peng 3,4, Jianguo Chen 3,4, Xueming Liu 5, Lianwu Fu 1,3, Tim M Townes 1, Steven M Rowe 3,4, David M Bedwell 1,3, Jennifer Guimbellot 2,3, Rui Zhao 1,3,*
Editor: Alessio Branchini6
PMCID: PMC10686454  PMID: 38019847

Abstract

A major unmet need in the cystic fibrosis (CF) therapeutic landscape is the lack of effective treatments for nonsense CFTR mutations, which affect approximately 10% of CF patients. Correction of nonsense CFTR mutations via genomic editing represents a promising therapeutic approach. In this study, we tested whether prime editing, a novel CRISPR-based genomic editing method, can be a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. We further demonstrated that prime editing may directly repair mutations in iPSC-derived airway epithelial cells when the prime editing machinery is efficiently delivered by helper-dependent adenovirus (HDAd). Together, our data demonstrated that prime editing may potentially be applied to correct CFTR mutations such as W1282X.

Introduction

Cystic fibrosis (CF) is caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a phosphorylation-regulated chloride/bicarbonate channel localized on the apical epithelial surface of the pulmonary and gastrointestinal tracts, pancreatic ducts, and the male reproductive ducts [1, 2]. Among the 2,000 mutations and genetic variations of CFTR, approximately 300 are disease-causing [3]. F508del, the most prevalent CFTR mutation, accounts for 70% of all mutant alleles. Nonsense CFTR mutations affect approximately 10% of all CF patients [4]. Tremendous progress has been made in developing small-molecule drugs to treat CF. Ivacaftor (VX-770), a drug that potentiates wild-type CFTR function and mutants such as the G551D with gating defects, can benefit approximately 5% of CF patients [5–8]. Trikafta, which combines ivacaftor and two corrector drugs elexacaftor/tezacaftor (i.e., VX-445/VX-661) can benefit patients with the F508del mutation [9–14]. Despite all the progress, treatments for nonsense CFTR mutations are currently unavailable.

Correction of nonsense CFTR mutations via genomic editing represents a promising approach toward disease amelioration and improved quality of life for patients. Conventional genomic editing methods, such as CRISPR/Cas9-mediated homology-directed repair (HDR), rely on the generation of DNA double-strand breaks (DSBs) at target genes, which often introduce unwanted de novo mutations (insertions/deletions (INDELs)) and chromosomal rearrangements [15–19]. Furthermore, HDR requires the use of a DNA correction template in addition to the guide RNA (gRNA) and Cas9 enzyme, which makes the in vivo delivery of the HDR repair machinery significantly more challenging. These difficulties could potentially be circumvented by prime editing, a novel CRISPR-based genomic editing approach [20]. Prime editing is mediated by a prime editing guide RNA (pegRNA) and a prime editor, which is a fusion protein consisting of a Cas9 nickase (nCas9) and a reverse transcriptase (RT) [20]. To achieve gene correction via prime editing, the pegRNA first trafficks the prime editor near the target site to nick one strand of the genomic DNA, the RT then reverse transcribes an RNA template (with corrected genetic information) embedded in the pegRNA to make a short corrected DNA strand, and this short DNA strand is incorporated into genomic DNA by cellular DNA repair machinery to achieve the correction [20]. Thus, prime editing-mediated gene correction does not require the delivery of a separate DNA repair template. Because of not introducing DSBs, prime editing also minimizes the risk of introducing INDELs. Prime editing has been shown to generate perfect nucleotide replacement up to 80 base pairs [20].

The inefficient delivery of gene correction machinery often represents a major challenge for gene therapy. Due to the large size, it took 2 to 3 lentivirus or adeno-associated viruses (AAVs) to deliver the prime editing machinery into cells [20]. Helper-dependent adenovirus (HDAd) is a promising delivery vehicle in gene therapy with a packaging capacity of up to 37 kb [21], which is sufficient to deliver the entire prime editing machinery. Importantly, HDAd has been demonstrated to efficiently transduce airway cells in cell cultures and animal models [22].

Patient cell-derived induced pluripotent stem cells (iPSCs), which carry all disease-causing mutations and retain the potential to differentiate into every adult cell type, have been considered an alternative human tissue source to model diseases, discover drugs, and develop cell and gene therapies [23]. In this study, we tested whether prime editing can be applied as a potential therapeutic modality to correct nonsense CFTR mutations. We generated iPSCs from a CF patient homozygous for the CFTR W1282X mutation. We demonstrated that prime editing corrected one mutant allele in iPSCs, which effectively restored CFTR function in iPSC-derived airway epithelial cells and organoids. Furthermore, we demonstrated that iPSC-derived airway epithelial cells may be edited when the prime editing machinery is delivered by HDAd. Together, our study demonstrated that prime editing may be applied to correct CFTR mutations such as W1282X.

Materials and methods

Ethics statement

Human nasal epithelial (HNE) cells were acquired as described with approval from the IRB of the University of Alabama at Birmingham (Protocol # IRB-151030001) [24]. Written informed consent, obtained from patients or guardians, was documented, and witnessed by a trained research coordinator.

Cell culture

HEK 293T cells were obtained from American Type Culture Collection (ATCC). HEK 293T cells were cultured in DMEM (Gibco) with 10% fetal bovine serum (FBS; GeminiBio). iPSCs were maintained and expanded on cell culture plates coated with hESC-qualified Geltrex basement membrane matrix (Thermo Scientific) in mTeSR medium (StemCell Technologies) as described [25].

iPSC generation and differentiation

iPSCs were generated as described [26]. In brief, 1 x 105 HNE W1282X cells were transduced with the STEMCCA lentivirus [27] and cultured in BEGM supplemented with 10 μM Y-27632 (Cayman Chemicals) for 7 days. The cells were then cultured in the E7 medium for 10 days followed by in the E8 medium for 12 days [28]. Single iPSC colonies were selected and expanded in mTeSR medium (StemCell Technologies) on cell culture plates coated with hESC-qualified Geltrex (Thermo Scientific). iPSCs were differentiated into airway epithelial cells and lung organoids by following the published protocol [29, 30]. In brief, iPSCs were cultured in STEMdiff Definitive Endoderm medium (StemCell Technologies) for 3 days, cSFDM supplemented with 10 μM SB431542 and 2 μM Dorsomorphin (Cayman Chemicals) for 4 days, and cSFDM supplemented with 3 μM CHIR99021 (Cayman Chemicals), 10 ng/mL recombinant human FGF10 (R&D Systems), 10 ng/mL recombinant human KGF (R&D Systems), 10 ng/mL recombinant human BMP4 (PeproTech), 50 nM retinoic acid (Millipore Sigma) for 11 more days. The cells were then mechanically dissociated into cell aggregates, embedded into growth factor-reduced Matrigel drops (50–100 μL in size), and cultured in the cSFDM medium supplemented with 250 ng/mL recombinant human FGF2 (ReproCell), 10 ng/mL FGF10 (R&D Systems), 50 nM dexamethasone (Millipore Sigma), 0.1 mM 8-Bromo-cyclic AMP sodium salt (Cayman Chemicals), 0.1 mM 3-Isobutyl-1-methyl-xanthine (IBMX) (Cayman Chemicals), and 10 μM Y-27632 (Cayman Chemicals) for 7 to 10 days. These cells were used for the differentiation of both airway epithelial cells and lung organoids.

For airway epithelial cell differentiation, single cells digested by Accutase (Thermo Scientific) were plated at 1 x105 cells/transwell insert of a 24-well plate (Corning), and cultured in PneumaCult-ALI maintenance medium (PAMM, StemCell Technologies) supplemented with 10 μM SB431542, 2 μM Dorsomorphin, and 10 μM Y-27632 for 7 to 12 days until reaching confluency. For air-liquid interface (ALI) culture, the cells were cultured for 2 to 4 weeks with no medium on the apical side. Media in the lower chambers were changed to PAMM medium.

For lung organoid differentiation, cells were dissociated into small aggregates by Accutase, embedded into growth factor-reduced Matrigel drops (50–100 μL in size) (Corning), and cultured in cSFDM medium supplemented with 250 ng/mL FGF2 and 10 ng/mL FGF10 for 7 to 14 days.

Prime editing vector construction

DNA oligonucleotides for pegRNA and nicking sgRNA (Integrated DNA Technologies) were cloned in pU6-pegRNA-GG-acceptor (Addgene #132777) by Golden Gate assembly (NEB) or the ligation method as described [20]. Sequences of pegRNA and nick RNA are listed in S1 Table in S1 Appendix. PE2 from the pCMV-PE2 plasmid (Addgene #132775) was inserted in pCL195, which drives the PE2-P2A-Puro expression cassette by an EF1α promoter. The pegRNA and nick sgRNA expression cassette was then transferred to pCL195 by Gibson assembly (NEB) to create the all-in-one pCL195-N-X1282W vector. All vectors were purified using QIAprep Spin Miniprep kits (QIAGEN).

Prime editing and sib selection

Two million iPSCs were dissociated into single cells by Accutase, washed with DMEM/F12 medium (Gibco), and resuspended in 100 μl Nucleofector solution (Lonza, VPH-5012). 6 μg pCL195-N-X1282W were added into iPSCs containing nucleofector solution, gently mixed, and nucleofected by the A-23 program of a Lonza 2B nucleofector (Lonza). A portion of the cells was directly plated onto a 96-well plate at ~ 50 cells/well for sib selection and the rest cells were plated onto one well of a 6-well plate. All cells were cultured in the mTeSR1 plus medium (StemCell Technologies) supplemented with 10 μM ROCK inhibitor Y-27632 (EMD Millipore). 0.375 μg/ml Puromycin was added to the medium 24 hrs later and maintained for 2 days. Genomic DNA samples were prepared by the DNeasy Blood & Tissue Kit (QIAGEN) for ddPCR analyses to estimate editing efficiency. Edited iPSC clones were isolated by sib selection as described [31]. In brief, the cells in the 96-well plate were split into two 96-well plates when the cell reached 80% confluency. One plate was used for genomic DNA extraction and the other plate for continuous cell culture. Genomic DNA was prepared by lysing cells with 25 μl prepGEM Universal (MicroGEM). 1 μl of DNA lysate was used as the template for ddPCR analyses. Wells with the highest gene correction efficiencies were selected for the next round of sib-selection until iPSC clones were isolated.

Droplet digital PCR (ddPCR) analysis

ddPCR was performed to examine the gene correction efficiency as described [19, 31]. In brief, each reaction contained 11 μl 2x ddPCR Supermix for Probes (Bio-Rad), 2 μl CCR5 primer mix (20 μM), 1 μl CCR5 TaqMan Probe (HEX, 5 μM), 2 μl target primer mix (20 μM), 1 μl target TaqMan Probe (FAM, 5 μM), 100 ng genomic DNA, and water to a final volume of 22 μl. Droplets were prepared by a QX200 Droplet Generator per the manufacturer’s instruction (Bio-Rad). PCR reactions were conducted on a Mastercycler Nexus PCR Thermal Cycler (Eppendorf) using a program consisting of 40 cycles of denaturation (95°C for 30 seconds) and annealing/elongation (57°C for 1 minute). The droplets were detected and analyzed by a QX200 Droplet Reader (Bio-Rad). Sequences of all primers and probes are listed in S1 Table in S1 Appendix.

PCR and genotype confirmation by Sanger sequencing

Genomic DNA purified by the DNeasy Blood & Tissue Kit (QIAGEN) was used as the DNA template for PCR reactions to amplify the DNA fragment containing the W1282 codon. The PCR product was purified by DNA Clean & Concentrator Kit (Zymo) before being sequenced using the forward primer. Primer sequences are listed in S1 Table in S1 Appendix.

Ussing chamber assay

Ussing chamber assay was performed as described [24, 32]. In brief, transwell inserts with iPSC-derived airway epithelial monolayers were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA) and short-circuit currents (Isc) were measured. The cells were initially bathed with Ringers solutions on both sides. After the suppression of the epithelial sodium channel (ENaC) by amiloride (100 μM, apical), the solution on the apical side was replaced by Ringers solution with low chloride to create a chloride gradient as described [32]. Reagents introduced sequentially to the bath solution include 100 μM Amiloride (apical), 10 μM forskolin (apical and basal), 10 μM VX-770 (apical and basal), 10 μM CFTRinh-172 (apical), and 20 μM GlyH101 (apical). Electrophysiological data were analyzed using Acquire and Analyze 2.3 software (Physiologic Instruments, San Diego, CA). All chemicals were purchased from Cayman Chemicals.

Organoid swelling assay

The organoid swelling assay was performed as described [24]. In brief, lung organoids were seeded onto μ-slide (15-well glass bottom slide, ibidi USA) at a density of ~ 25 organoids/well and cultured in cSFDM medium supplemented with 250 ng/mL FGF2 and 10 ng/mL FGF10 overnight. Right before forskolin stimulation and imaging, the organoids were pre-incubated with NucBlu (Thermo Scientific) for 1 hour. After adding 10 μM forskolin, organoids were imaged every 20 min for 8 hours via the bright field and DAPI fluorescent channels using a Lionheart FX automated imaging system (BioTek). Image processing and the automated quantification of organoid sizes over time were achieved by using the Gen5 ImagePrime software (BioTek).

Helper-dependent adenoviral (HDAd) vector construction, virus preparation, and HDAd-mediated prime editing

The W1282X correction HDAd vector pCL196 was constructed by transferring the prime editing components from the all-in-one pCL195-N-X1282W vector to pHDAd-26K-GW, an HDAd vector backbone derived from pDelta28ElacZ [21], via I-CeuI/I-SceI. 10 μg of recombinant adenoviral plasmids were digested with PmeI and packaged into HDAd virus by using the helper virus rADNG163 in 116 cells (gifts from Dr. Philip Ng) as described [21, 33]. The titer of the rCL196 HDAd virus was determined by the Endpoint-Dilution Assay [34]. W1282X iPSC-derived airway epithelial cells cultured in ALI culture for 2–3 weeks were used for prime editing. Viral transduction was conducted by adding the rCL196 HDAd virus (an estimated MOI of 100) to the bottom chamber of the transwell. Genomic DNA samples were collected 96 hours after viral transduction and were used for ddPCR analyses to determine editing efficiencies.

Immunofluorescence and microscopy

Immunofluorescence was conducted as described [35]. In brief, cells were fixed in 4% paraformaldehyde, blocked in Protein Block (Agilent), and incubated with the appropriate primary antibodies overnight at 4°C and secondary antibodies for 1 hr at room temperature. Nuclei were counterstained by 0.5 μg/mL DAPI. F-Actin was stained by phalloidin conjugated with Alexa Fluor 568 (Thermo Scientific). Images were acquired by a Nikon Ti-S or a Nikon A1R-HD25 confocal microscope and processed by Elements AR software (Nikon). Antibodies used were as followings: SOX2 (09–0024, ReproCell), FOXA2 (sc-101060, Santa Cruz Biotech), NKX2.1 (ab76013, Abcam), ZO-1 (MA3-39100-A647, Thermo Scientific), MUC5B (HPA008246, Sigma-Aldrich), α-Tubulin (ab11315, Abcam), CFTR (MAB25031, R&D).

Results

CFTR W1282X mutation is amenable to prime editing-mediated gene correction

We analyzed the two most common nonsense CFTR mutations (i.e., G542X and W1282X) and found the sequence surrounding the W1282X mutation enabled the design of a pegRNA for prime editing (Fig 1A). To experimentally validate that prime editing can correct the W1282X mutation, we first designed a W1282-pegRNA that enables conversion of the wild-type TGG codon encoding the W1282 residue to a TGA stop codon in HEK 293T cells (S1A Fig in S1 Appendix). HEK 293T cells were used to test and optimize prime editing strategies primarily because they are easy to culture and transfect as shown in previous studies [19, 36, 37].

Fig 1. CFTR W1282X mutation is amenable to prime editing-mediated gene correction.

Fig 1

Open in a new tab

(A) The W1282X-pegRNA and the DNA target. PBS, primer binding site. In addition to the tracrRNA (solid blue line) that binds to the nCas9, each pegRNA contains three key functional components–the crRNA, PBS, and template. crRNA brings the prime editor to the target site. PBS, primer binding site. (B) Generation of W1282X-iPSCs from nasal epithelial cells of a CF patient with homozygous CFTR W1282X mutations. (C) Sequencing analyses of PCR fragments containing the W1282 codon amplified from genomic DNA of W1282X-iPSCs (top) and PE-iPSCs (bottom). Blue box, the W1282X codon. (D) Droplet digital PCR (ddPCR) analyses indicated that transfection of prime editing machinery to W1282X-iPSCs corrected 0.36% of mutant alleles. Negative control, W1282X-iPSCs not transfected with prime editing machinery. The W1282 probe only recognizes corrected CFTR alleles. The CCR5 probe is used as a copy number control for the input genomic DNA.

It has been shown that introducing a nick on the non-edited DNA strand by nCas9 may enhance prime editing by facilitating the incorporation of the edited strand during DNA repair [20]. DNA sequence analysis identified two potential nCas9 cutting sites on the non-edited strand (S1B Fig in S1 Appendix). By co-transfecting plasmids expressing the prime editor, W1282-pegRNA, and a nick gRNA, we tested how each of the two nick sites affected the editing efficiency of the W1282 codon in HEK 293T cells. To determine editing efficiency, we developed droplet digital PCR (ddPCR) assays that can specifically detect the W1282X mutant allele. The ddPCR analysis showed that nick gRNA(-37)-1 led to an approximately 2-fold increase in editing efficiency on the W1282 codon (S1C Fig in S1 Appendix). Based on these data, the nick gRNA(-37)-1 was used for the rest of the study.

The lengths of the primer binding sequence (PBS) and the template of reverse transcription on pegRNAs have also been suggested to impact the efficiency of prime editing in a locus-dependent manner [20]. We then tested editing efficiencies on the W1282 codon by pegRNAs with different PBS and RT template lengths (S1D, S1E Fig in S1 Appendix). We found that pegRNAs with an 11-nucleotide (nt) PBS and a 15-nt RT template were more efficient in editing the W1282 codon (S1F Fig in S1 Appendix). Based on these data, we chose the P11T15 pegRNA, which contains an 11-nt PBS and a 15-nt RT template, to correct the W1282X mutation (Fig 1A).

To test whether prime editing can correct the W1282X mutation and restore CFTR function, we generated iPSCs from a patient homozygous for the CFTR W1282X mutation (Fig 1B and 1C). We designed a W1282X-pegRNA targeting the DNA sequence encoding the W1282X mutation (Fig 1A) and transfected the plasmid co-expressing the W1282X-pegRNA, prime editor, and nick gRNA(-37)-1 to W1282X-iPSCs. The corrected CFTR alleles can be detected at a frequency of 0.36% by ddPCR analyses, which distinguish the corrected W1282-encoding DNA sequence from the W1282X mutant DNA sequence (Fig 1D). We then performed sib selection to isolate iPSCs carrying prime-editing corrected CFTR alleles (PE-iPSCs). We observed a ~10-fold enrichment (0.36% to 3.4%) after one round of selection (S2A Fig in S1 Appendix) and isolated four independent PE-iPSC clones. Sanger sequencing analyses confirmed that all the PE-iPSC clones contained one repaired CFTR allele (Fig 1C and S2B Fig in S1 Appendix). Furthermore, INDELs were observed on neither the repaired nor the unrepaired CFTR allele in any of the clones (S2B Fig in S1 Appendix).

Differentiation of iPSCs to airway epithelial cells and lung organoids

Previous studies have demonstrated that iPSC-derived airway epithelial cells and lung organoids may be used to test CFTR function in Ussing chamber assays and forskolin-induced swelling assays [29, 38]. To test whether prime editing-mediated gene correction had restored CFTR function, we differentiated iPSCs into airway epithelial cells and lung organoids using the published protocol [29]. iPSC differentiation involves recapitulation of embryonic lung development in vivo, which undergoes a series of lineage specification events including definitive endoderm (DE), anterior foregut, lung progenitor cells, and lung epithelium (Fig 2A). We confirmed that the anterior foregut cells expressed the stage-specific transcriptional factors SOX2 and FOXA2 (Fig 2B–2B’) and the lung progenitors expressed the stage-specific markers NKX2.1 and FOXA2 (Fig 2C). We also confirmed that the iPSC-derived airway epithelial cells expressed ZO-1, the marker for tight junction, and contained mucin-producing (MUC5B+) and multiciliated (acetylated tubulin+) cells (Fig 2D and 2E). Confocal microscopy further confirmed the apical expression of CFTR in a fraction of airway epithelial cells (Fig 2F).

Fig 2. Differentiation of iPSCs to airway epithelial cells and lung organoids.

Fig 2

Open in a new tab

(A) Schematic of the iPSC differentiation protocol to airway epithelial cells and lung organoids. DE, definitive endoderm. ALI, air-liquid interface. (B—F) Immunofluorescence analyses of stage-specific markers and lung epithelial cell markers. (B-B’) Anterior foregut cells express stage-specific markers (B) SOX2 and (B’) FOXA2. (C) Lung progenitor cells express stage-specific markers NKX2.1 and FOXA2. (D-F) iPSC-derived lung epithelium (D, E) expresses the tight junction marker ZO-1 and contains cells positive for (D) MUB5B, (E) acetylated tubulin, and (F) CFTR. Note that CFTR is expressed on the apical side (XZ and YZ) of the epithelium. White scale bars, 100 μm. Red scale bar, 20 μm.

Prime editing restored CFTR function in PE-iPSC-derived lung tissues

To test whether prime editing had restored CFTR function, we performed Ussing chamber assays and forskolin-induced swelling (FIS) assays using airway epithelial cells and lung organoids from prime editing-corrected iPSCs (PE-iPSCs) (Fig 3). In the Ussing chamber assays, airway epithelial cells differentiated from PE-iPSCs showed significant activation of short circuit current (Isc) after forskolin (FSK) treatment, which can be further enhanced when treating with VX-770, the potentiator CFTR drug showing pronounced efficacies to mutants with gating defects. The change of Isc depends on the CFTR function because the CFTR inhibitor-172 and GlyH101 can nearly completely suppress the FSK and VX-770-induced current changes (Fig 3A and 3B). In contrast, airway epithelial cells differentiated from W1282X-iPSCs showed responses to neither FSK nor VX-770, supporting that the W1282X mutant is not functional (Fig 3A and 3B). In the FIS assays, lung organoids differentiated from PE-iPSCs swelled upon FSK stimulation over an 8-hour period, while lung organoids differentiated from W1282X-iPSCs exhibited little responses to FSK (Fig 3C and 3D, and supporting movies). Together, these data demonstrated that prime editing-mediated gene correction had restored CFTR function.

Fig 3. Prime editing restored CFTR function in PE-iPSC-derived lung tissues.

Fig 3

Open in a new tab

(A) Ussing chamber assays that measure short circuit current (Isc) traces of airway epithelial differentiated from W1282X-iPSCs and PE-iPSCs. Amil, amiloride; LoCl, low chloride solution; FSK, forskolin; INH 172, CFTR Inhibitor-172; n = 3 biological repeats. (B) Quantification of the (A). Error bars, SD. (C) Forskolin-induced swelling assays of lung organoids differentiated from W1282X-iPSCs and PE-iPSCs. Shown are images of representative wells before (0 min) and after (480 min) forskolin induction. (D) Quantification of (C) was recorded in five independent wells. Error bars, SD.

Prime editing corrects the W1282X mutation in differentiated airway epithelial cells

To become a potential therapeutic modality, prime editing must correct CFTR mutations in differentiated airway epithelial cells but not in undifferentiated iPSCs. Delivery of plasmids that express prime editing machinery to airway epithelial cells by nucleofection and related transfection methods had proven ineffective. To efficiently deliver the prime editing machinery and better mimic a therapeutic setting, we constructed an all-in-one helper-dependent adenovirus (HDAd) that expresses all the necessary components of prime editing, which includes the prime editor, the W1282X-pegRNA, the nick gRNA(-37)-1, and a GFP reporter (Fig 4A). Our data showed that HDAd can achieve 71.7 ± 5.4% transduction efficiency in iPSC-derived airway epithelial cells, as monitored by GFP expression (S3 Fig in S1 Appendix). ddPCR analyses demonstrated that 2.4 ± 0.6% of W1282X mutant alleles had been corrected by HDAd-delivered prime editing (Fig 4B and 4C). In the Ussing chamber assays, a minor increase of Isc was observed following FSK stimulation in edited airway epithelia, whereas this response is absent in the non-edited controls (S4A Fig in S1 Appendix). Comparing this to the PE-iPSC-derived airway epithelium (Fig 3B), the amplitude of the FSK-induced Isc is approximately 20-fold lower (S4B Fig in S1 Appendix), which is in line with the expected functional CFTR allele frequencies (~2.5% vs. 50%). However, neither the edited nor non-edited airway epithelia exhibited an increase in Isc upon VX-770 stimulation (S4 Fig in S1 Appendix), possibly due to the low Isc signals or other unidentified factors.

Fig 4. Prime editing corrects the W1282X mutation in differentiated airway epithelial cells.

Fig 4

Open in a new tab

(A) Schematic of the all-in-one HDAd vector. (B) ddPCR analyses to measure the correction efficiency in HDAd-transduced airway epithelial cells. Negative control, cells not transduced with HDAd. The W1282 probe only recognizes corrected CFTR alleles. The CCR5 probe is used as a copy number control for the input genomic DNA. n = 7 biological repeats. (C) Quantification of (B). Error bars, SD.

Discussion

Lung diseases are the leading cause of morbidity and mortality in CF patients [1]. Because modeling lung disease requires the use of healthy and diseased human lung epithelial cells, the shortage of human lung tissue hinders CF research and drug discovery. CF patient-derived iPSCs, which carry the disease-causing CFTR mutations, have been used to model the disease, discover drug candidates, and test gene therapy strategies. Several laboratories have developed protocols to differentiate iPSCs into lung tissues [29, 30, 39–46]. The iPSC-derived airway epithelium and lung organoids form tight junctions, express CFTR, and can be used to assay CFTR channel functions by electrophysiological (e.g., whole-cell patch clamp and Ussing chamber assays) and FIS assays [29, 30, 38, 44]. CF patient-derived iPSCs and the derived airway epithelial cells have also been used to test the various genomic editing approaches, including CRISPR/Cas9-mediated HDR [29, 44].

In this study, we demonstrated that prime editing may restore the function of the CFTR W1282X mutant. Compared to the conventional CRISPR/Cas9-mediated HDR, prime editing does not generate DSBs, which minimizes the likelihood of introducing INDELs and chromosomal rearrangements. Because prime editing does not rely on homologous recombination, it can edit cells with low proliferative potentials, such as airway epithelial cells (Fig 4). In addition, unlike CRISPR/Cas9-mediated HDR, prime editing does not require a separate repair DNA template. The template module of pegRNA contains the necessary genetic information for gene correction, which is reverse transcribed and incorporated into the genome. Without inducing DSBs and introducing exogenous repair DNA templates, prime editing is likely less toxic to cells.

In this study, we were able to design a pegRNA to correct the CFTR W1282X mutation but not the G542X mutation using the currently available prime editor PE2. Because PE2 is derived from the conventional Cas9 protein, which strictly requires the NGG PAM sequence for target recognition [20]. Because Cas9 variants recognizing a broader range of PAM sequences, such as xCas9 or Cas9-NG, had been derived [47, 48], more versatile prime editors that would edit a much larger scope of genomic targets may be developed in future studies to edit genomic targets such as the CFTR G542X mutation.

While our data demonstrated prime editing can correct the CFTR W1282X mutation in iPSCs and airway epithelial cells, the correction efficiency remains low. The overall correction efficiency is affected by the delivery efficiency of the editing machinery to cells and the editing efficiency of the DNA target. To achieve high delivery efficiency into airway epithelial cells, we used HDAd. Adenovirus is a commonly used delivery vehicle in gene therapy because of its well-characterized biology, broad tropism, and large cloning capacity. Compared to conventional adenovirus, HDAd is less toxic to cells because of the removal of all viral coding sequences [21, 33]. Our data showed that while the great majority of the airway epithelial cells (~ 70%) had received the prime editing machinery (S3 Fig in S1 Appendix), only a fraction (~ 2.5%) of DNA targets were corrected (Fig 4B and 4C). If considering most cells contained a heterozygous repair, the total repaired airway epithelial cells would likely be between 2.5–5%. The editing efficiency of prime editing is locus-dependent. The editing efficiency could be up to 80% at some genetic loci but low at others [49]. Therefore, a better understanding of the variables affecting the editing efficiency, which would lead to further optimization of the prime editing machinery, will be essential to apply prime editing in correcting mutations such as CFTR W1282X.

The restoration of 10 to 35% of CFTR activity has been estimated as a necessary threshold to alleviate pulmonary morbidity [50]. Consequently, achieving a correction level spanning from 10 to 35% of mutant CFTR alleles is likely imperative for effective CF treatments. However, if the corrected cells primarily comprise basal cells, which can repopulate the airway with functional CFTR-carrying epithelial cells lower editing efficiency may still yield therapeutic benefits. HDAd has proven effective in transducing airway basal cells in animal models [22]. Therefore, gaining a deeper understanding of the specific cell types corrected by prime editing would yield valuable insights into the prospective applications of gene therapy for CF treatment.

Recently, base editing, a novel CRISPR-based genomic editing method that enables an A to G or a C to T transition without introducing DSBs and INDELs [51, 52], had been successfully applied to repair three CFTR mutations including the W1282X mutation [53]. The editing efficiency of the W1282X codon can achieve 69% in cultured cells, which makes base editing among the most promising gene therapy methods. However, base editing generates off-target base substitutions in the DNA genome and particularly on RNA molecules, primarily because base editors can modify any DNA or RNA substrates in proximity [54–58]. The A residue immediately downstream of the CFTR W1282X codon had been converted to a G residue in 16% of the genome, which led to a de novo arginine to glycine (AGG → GGG) substitution [53]. Compared to base editing, prime editing shows little off-target effects because prime editors only function after sequence complementations between the DNA target and both gRNA and PBS (Fig 1A). However, likely contributed by the additional demand for sequence complementation, prime editing is generally less efficient, and relatively fewer genomic loci are amenable to its modification. Very recently, the CFTR F508del mutation had been repaired by prime editing in human intestinal organoids but also at low efficiency [59]. Several recent studies suggested that the efficiency of prime editing may be significantly improved. Nelson et al. reported pegRNAs may be stabilized by adding a structured RNA motif to the 3’ ends, which led to a 3 to 4-fold increase in the editing efficiencies of tested loci [60]. Chen et al. reported a 3 to 7-fold increase in prime editing efficiency may be achieved by manipulating the cellular DNA mismatch repair pathway [61]. Anzalone et al. reported a twin prime editing approach, which involves a pair of pegRNAs simultaneously editing two adjacent sites surrounding the targeting locus, could not only enable deletion, replacement, and insertion of larger DNA fragments but also enhance editing efficiency [49]. Together with our results, prime editing may be a strategy to correct CFTR mutations, however, further optimization to improve editing efficiency is required.

Supporting information

S1 Appendix. It includes four supporting figures, figure legends, and a supporting table.

(DOCX)

Click here for additional data file. (2.6MB, docx)
S2 Appendix. W1282X organoid FIS assay.

(WMV)

Click here for additional data file. (5.5MB, wmv)
S3 Appendix. PE organoid FIS assay.

(WMV)

Click here for additional data file. (5.4MB, wmv)

Acknowledgments

We thank Dr. Philip Ng for kindly providing adenoviral plasmid, helper virus, and the packing cell line.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med. 2005;352(19):1992–2001. doi: 10.1056/NEJMra043184 . [DOI] [PubMed] [Google Scholar]
  • 2.Ratjen F, Bell SC, Rowe SM, Goss CH, Quittner AL, Bush A. Cystic fibrosis. Nat Rev Dis Primers. 2015;1:15010. Epub 2015/01/01. doi: 10.1038/nrdp.2015.10 . [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Castellani C, Cuppens H, Macek M Jr., Cassiman JJ, Kerem E, Durie P, et al. Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice. J Cyst Fibros. 2008;7(3):179–96. Epub 2008/05/06. doi: 10.1016/j.jcf.2008.03.009 ; PubMed Central PMCID: PMC2810954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Bobadilla JL, Macek M Jr., Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations—correlation with incidence data and application to screening. Hum Mutat. 2002;19(6):575–606. Epub 2002/05/15. doi: 10.1002/humu.10041 . [DOI] [PubMed] [Google Scholar]
  • 5.Kotha K, Clancy JP. Ivacaftor treatment of cystic fibrosis patients with the G551D mutation: a review of the evidence. Ther Adv Respir Dis. 2013;7(5):288–96. Epub 2013/09/06. doi: 10.1177/1753465813502115 . [DOI] [PubMed] [Google Scholar]
  • 6.Kapoor H, Koolwal A, Singh A. Ivacaftor: a novel mutation modulating drug. J Clin Diagn Res. 2014;8(11):SE01–5. Epub 2015/01/15. doi: 10.7860/JCDR/2014/6486.5158 ; PubMed Central PMCID: PMC4290359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Molloy K, McElvaney NG. Ivacaftor: from bench to bedside… and back again. Am J Respir Crit Care Med. 2014;190(2):128–9. Epub 2014/07/16. doi: 10.1164/rccm.201406-1122ED . [DOI] [PubMed] [Google Scholar]
  • 8.Wainwright CE. Ivacaftor for patients with cystic fibrosis. Expert Rev Respir Med. 2014;8(5):533–8. Epub 2014/08/26. doi: 10.1586/17476348.2014.951333 . [DOI] [PubMed] [Google Scholar]
  • 9.Taylor-Cousar JL, Munck A, McKone EF, van der Ent CK, Moeller A, Simard C, et al. Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del. N Engl J Med. 2017;377(21):2013–23. Epub 2017/11/04. doi: 10.1056/NEJMoa1709846 . [DOI] [PubMed] [Google Scholar]
  • 10.Rowe SM, Daines C, Ringshausen FC, Kerem E, Wilson J, Tullis E, et al. Tezacaftor-Ivacaftor in Residual-Function Heterozygotes with Cystic Fibrosis. N Engl J Med. 2017;377(21):2024–35. Epub 2017/11/04. doi: 10.1056/NEJMoa1709847 ; PubMed Central PMCID: PMC6472479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Holguin F. Triple CFTR Modulator Therapy for Cystic Fibrosis. N Engl J Med. 2018;379(17):1671–2. Epub 2018/10/20. doi: 10.1056/NEJMe1811996 . [DOI] [PubMed] [Google Scholar]
  • 12.Keating D, Marigowda G, Burr L, Daines C, Mall MA, McKone EF, et al. VX-445-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles. N Engl J Med. 2018;379(17):1612–20. Epub 2018/10/20. doi: 10.1056/NEJMoa1807120 ; PubMed Central PMCID: PMC6289290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Davies JC, Moskowitz SM, Brown C, Horsley A, Mall MA, McKone EF, et al. VX-659-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles. N Engl J Med. 2018;379(17):1599–611. Epub 2018/10/20. doi: 10.1056/NEJMoa1807119 ; PubMed Central PMCID: PMC6277022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Zemanick ET, Taylor-Cousar JL, Davies J, Gibson RL, Mall MA, McKone EF, et al. A Phase 3 Open-Label Study of Elexacaftor/Tezacaftor/Ivacaftor in Children 6 through 11 Years of Age with Cystic Fibrosis and at Least One F508del Allele. Am J Respir Crit Care Med. 2021;203(12):1522–32. Epub 2021/03/19. doi: 10.1164/rccm.202102-0509OC ; PubMed Central PMCID: PMC8483230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wright AV, Nunez JK, Doudna JA. Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering. Cell. 2016;164(1–2):29–44. Epub 2016/01/16. doi: 10.1016/j.cell.2015.12.035 . [DOI] [PubMed] [Google Scholar]
  • 16.Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765–71. Epub 2018/07/17. doi: 10.1038/nbt.4192 ; PubMed Central PMCID: PMC6390938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cullot G, Boutin J, Toutain J, Prat F, Pennamen P, Rooryck C, et al. CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun. 2019;10(1):1136. Epub 2019/03/10. doi: 10.1038/s41467-019-09006-2 ; PubMed Central PMCID: PMC6408493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24(7):927–30. Epub 2018/06/13. doi: 10.1038/s41591-018-0049-z . [DOI] [PubMed] [Google Scholar]
  • 19.Li C, Liu Z, Zhang X, Wang H, Friedman GK, Ding Q, et al. Generation of chromosome 1p/19q co-deletion by CRISPR/Cas9-guided genomic editing. Neuro-Oncology Advances. 2022. doi: 10.1093/noajnl/vdac131 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576(7785):149–57. Epub 2019/10/22. doi: 10.1038/s41586-019-1711-4 ; PubMed Central PMCID: PMC6907074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Palmer D, Ng P. Improved system for helper-dependent adenoviral vector production. Mol Ther. 2003;8(5):846–52. Epub 2003/11/06. doi: 10.1016/j.ymthe.2003.08.014 . [DOI] [PubMed] [Google Scholar]
  • 22.Cao H, Ouyang H, Grasemann H, Bartlett C, Du K, Duan R, et al. Transducing Airway Basal Cells with a Helper-Dependent Adenoviral Vector for Lung Gene Therapy. Hum Gene Ther. 2018;29(6):643–52. Epub 2018/01/13. doi: 10.1089/hum.2017.201 . [DOI] [PubMed] [Google Scholar]
  • 23.Robinton DA, Daley GQ. The promise of induced pluripotent stem cells in research and therapy. Nature. 2012;481(7381):295–305. Epub 2012/01/20. doi: 10.1038/nature10761 ; PubMed Central PMCID: PMC3652331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Liu Z, Anderson JD, Deng L, Mackay S, Bailey J, Kersh L, et al. Human Nasal Epithelial Organoids for Therapeutic Development in Cystic Fibrosis. Genes (Basel). 2020;11(6). Epub 2020/06/04. doi: 10.3390/genes11060603 ; PubMed Central PMCID: PMC7349680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Lee J, Liu Z, Tusing YG, Li C, Westin E, Li W, et al. Generation of inducible pluripotent stem cell lines from Alzheimer’s disease patients with APOE e3/e3 genotype. Stem Cell Res. 2021;55:102498. Epub 2021/08/16. doi: 10.1016/j.scr.2021.102498 ; PubMed Central PMCID: PMC8496340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Liu Z, Che P, Mercado JJ, Hackney JR, Friedman GK, Zhang C, et al. Characterization of iPSCs derived from low grade gliomas revealed early regional chromosomal amplifications during gliomagenesis. J Neurooncol. 2019;141(2):289–301. Epub 2018/11/22. doi: 10.1007/s11060-018-03047-1 ; PubMed Central PMCID: PMC6344247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Sommer CA, Stadtfeld M, Murphy GJ, Hochedlinger K, Kotton DN, Mostoslavsky G. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells. 2009;27(3):543–9. Epub 2008/12/20. doi: 10.1634/stemcells.2008-1075 ; PubMed Central PMCID: PMC4848035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424–9. Epub 2011/04/12. doi: 10.1038/nmeth.1593 ; PubMed Central PMCID: PMC3084903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.McCauley KB, Hawkins F, Serra M, Thomas DC, Jacob A, Kotton DN. Efficient Derivation of Functional Human Airway Epithelium from Pluripotent Stem Cells via Temporal Regulation of Wnt Signaling. Cell Stem Cell. 2017;20(6):844–57 e6. Epub 2017/04/04. doi: 10.1016/j.stem.2017.03.001 ; PubMed Central PMCID: PMC5457392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Hawkins F, Kramer P, Jacob A, Driver I, Thomas DC, McCauley KB, et al. Prospective isolation of NKX2-1-expressing human lung progenitors derived from pluripotent stem cells. J Clin Invest. 2017;127(6):2277–94. Epub 2017/05/04. doi: 10.1172/JCI89950 ; PubMed Central PMCID: PMC5451263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li C, Ding L, Sun CW, Wu LC, Zhou D, Pawlik KM, et al. Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction. Sci Rep. 2016;6:30422. Epub 2016/07/28. doi: 10.1038/srep30422 ; PubMed Central PMCID: PMC4961958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Rowe SM, Pyle LC, Jurkevante A, Varga K, Collawn J, Sloane PA, et al. DeltaF508 CFTR processing correction and activity in polarized airway and non-airway cell monolayers. Pulm Pharmacol Ther. 2010;23(4):268–78. Epub 2010/03/17. doi: 10.1016/j.pupt.2010.02.001 ; PubMed Central PMCID: PMC2885545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Palmer DJ, Ng P. Physical and infectious titers of helper-dependent adenoviral vectors: a method of direct comparison to the adenovirus reference material. Mol Ther. 2004;10(4):792–8. Epub 2004/09/29. doi: 10.1016/j.ymthe.2004.06.1013 . [DOI] [PubMed] [Google Scholar]
  • 34.Lock M, Korn M, Wilson J, Sena-Esteves M, Gao G. Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID(50)) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR). Cold Spring Harb Protoc. 2019;2019(8). Epub 2019/08/03. doi: 10.1101/pdb.prot095562 . [DOI] [PubMed] [Google Scholar]
  • 35.Liu Z, Zhang C, Skamagki M, Khodadadi-Jamayran A, Zhang W, Kong D, et al. Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem Cells. Stem Cell Reports. 2017;9(5):1604–17. Epub 2017/11/16. doi: 10.1016/j.stemcr.2017.10.006 ; PubMed Central PMCID: PMC5688240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. Epub 2013/01/05. doi: 10.1126/science.1231143 ; PubMed Central PMCID: PMC3795411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. Epub 2013/01/05. doi: 10.1126/science.1232033 ; PubMed Central PMCID: PMC3712628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Crane AM, Kramer P, Bui JH, Chung WJ, Li XS, Gonzalez-Garay ML, et al. Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells. Stem Cell Reports. 2015;4(4):569–77. Epub 2015/03/17. doi: 10.1016/j.stemcr.2015.02.005 ; PubMed Central PMCID: PMC4400651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Green MD, Chen A, Nostro MC, d’Souza SL, Schaniel C, Lemischka IR, et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):267–72. Epub 2011/03/02. doi: 10.1038/nbt.1788 ; PubMed Central PMCID: PMC4866999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, et al. Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells. Cell Stem Cell. 2012;10(4):398–411. Epub 2012/04/10. doi: 10.1016/j.stem.2012.01.019 ; PubMed Central PMCID: PMC3322392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Mou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, et al. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell. 2012;10(4):385–97. doi: 10.1016/j.stem.2012.01.018 ; PubMed Central PMCID: PMC3474327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Wong AP, Bear CE, Chin S, Pasceri P, Thompson TO, Huan LJ, et al. Directed differentiation of human pluripotent stem cells into mature airway epithelia expressing functional CFTR protein. Nat Biotechnol. 2012;30(9):876–82. doi: 10.1038/nbt.2328 ; PubMed Central PMCID: PMC3994104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Huang SX, Islam MN, O’Neill J, Hu Z, Yang YG, Chen YW, et al. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol. 2014;32(1):84–91. doi: 10.1038/nbt.2754 ; PubMed Central PMCID: PMC4101921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Firth AL, Dargitz CT, Qualls SJ, Menon T, Wright R, Singer O, et al. Generation of multiciliated cells in functional airway epithelia from human induced pluripotent stem cells. Proc Natl Acad Sci U S A. 2014;111(17):E1723–30. doi: 10.1073/pnas.1403470111 ; PubMed Central PMCID: PMC4035971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Chen YW, Huang SX, de Carvalho A, Ho SH, Islam MN, Volpi S, et al. A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol. 2017;19(5):542–9. Epub 2017/04/25. doi: 10.1038/ncb3510 ; PubMed Central PMCID: PMC5777163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Konishi S, Gotoh S, Tateishi K, Yamamoto Y, Korogi Y, Nagasaki T, et al. Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells. Stem Cell Reports. 2016;6(1):18–25. Epub 2016/01/05. doi: 10.1016/j.stemcr.2015.11.010 ; PubMed Central PMCID: PMC4720023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7699):57–63. Epub 2018/03/08. doi: 10.1038/nature26155 ; PubMed Central PMCID: PMC5951633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Nishimasu H, Shi X, Ishiguro S, Gao L, Hirano S, Okazaki S, et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science. 2018;361(6408):1259–62. Epub 2018/09/01. doi: 10.1126/science.aas9129 ; PubMed Central PMCID: PMC6368452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Anzalone AV, Gao XD, Podracky CJ, Nelson AT, Koblan LW, Raguram A, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol. 2022;40(5):731–40. Epub 2021/12/11. doi: 10.1038/s41587-021-01133-w ; PubMed Central PMCID: PMC9117393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Kerem E. Pharmacologic therapy for stop mutations: how much CFTR activity is enough? Curr Opin Pulm Med. 2004;10(6):547–52. Epub 2004/10/29. doi: 10.1097/01.mcp.0000141247.22078.46 . [DOI] [PubMed] [Google Scholar]
  • 51.Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature. 2017;551(7681):464–71. Epub 2017/11/22. doi: 10.1038/nature24644 ; PubMed Central PMCID: PMC5726555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–4. Epub 2016/04/21. doi: 10.1038/nature17946 ; PubMed Central PMCID: PMC4873371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Krishnamurthy S, Traore S, Cooney AL, Brommel CM, Kulhankova K, Sinn PL, et al. Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors. Nucleic Acids Res. 2021;49(18):10558–72. Epub 2021/09/15. doi: 10.1093/nar/gkab788 ; PubMed Central PMCID: PMC8501978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Kang Y, Dai S, Zeng Y, Wang F, Yang P, Yang Z, et al. Cloning and base editing of GFP transgenic rhesus monkey and off-target analysis. Sci Adv. 2022;8(29):eabo3123. Epub 2022/07/23. doi: 10.1126/sciadv.abo3123 ; PubMed Central PMCID: PMC9307242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Zhou C, Sun Y, Yan R, Liu Y, Zuo E, Gu C, et al. Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature. 2019;571(7764):275–8. Epub 2019/06/11. doi: 10.1038/s41586-019-1314-0 . [DOI] [PubMed] [Google Scholar]
  • 56.Grunewald J, Zhou R, Garcia SP, Iyer S, Lareau CA, Aryee MJ, et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature. 2019;569(7756):433–7. Epub 2019/04/18. doi: 10.1038/s41586-019-1161-z ; PubMed Central PMCID: PMC6657343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science. 2019;364(6437):289–92. Epub 2019/03/02. doi: 10.1126/science.aav9973 ; PubMed Central PMCID: PMC7301308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science. 2019;364(6437):292–5. Epub 2019/03/02. doi: 10.1126/science.aaw7166 . [DOI] [PubMed] [Google Scholar]
  • 59.Geurts MH, de Poel E, Pleguezuelos-Manzano C, Oka R, Carrillo L, Andersson-Rolf A, et al. Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Sci Alliance. 2021;4(10). Epub 2021/08/11. doi: 10.26508/lsa.202000940 ; PubMed Central PMCID: PMC8356249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Nelson JW, Randolph PB, Shen SP, Everette KA, Chen PJ, Anzalone AV, et al. Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol. 2022;40(3):402–10. Epub 2021/10/06. doi: 10.1038/s41587-021-01039-7 ; PubMed Central PMCID: PMC8930418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Chen PJ, Hussmann JA, Yan J, Knipping F, Ravisankar P, Chen PF, et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell. 2021;184(22):5635–52 e29. Epub 2021/10/16. doi: 10.1016/j.cell.2021.09.018 ; PubMed Central PMCID: PMC8584034. [DOI] [PMC free article] [PubMed] [Google Scholar]

Decision Letter 0

Alessio Branchini

4 Sep 2023

PONE-D-23-20582

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

PLOS ONE

Dear Dr. Zhao,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Oct 19 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Alessio Branchini

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

   "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

   "R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594.

NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in the Competing Interests section: 

   "I have read the journal's policy and the authors of this manuscript have the following competing interests:

S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. "

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests).  If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. 

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: I Don't Know

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The study “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, by Li and colleagues is of high quality and of interest to a broad audience. The results presented in the main figures of the manuscript provide a concise and impactful study on the use of prime editing to “fix” CFTR mutation p.W1282X. The major advances presented here are the use of an “all-in-one” helper dependent Adenovirus (HDAd) to efficiently deliver all of the prime editing machinery to iPSC-derived airway epithelial cells and restore p.W1282X-CFTR function. Because of the size of the payload, this has previously not been able to be achieved. This reviewer found much of the interesting biology to be in the supplemental figures, where a good amount of optimization was performed. There are only a few comments to address.

Comments:

1) The authors touted the ability of delivering all of the machinery in a helper dependent adenovirus (HDAd) system for efficient editing. However, even with a plasmid system containing all of the PE machinery and high transfection efficiency, the editing efficiency was only 0.36% in iPSCs. (Fig 1D). With the HDAd system providing ~70% transduction efficiency of differentiated airway epithelial cells only corrected 2-3% of the p.W1282X alleles. Indeed, the bottleneck for correction of p.W1282X CF mutations is the overall efficiency of prime editing, not delivery. While there is nice discussion on this issue, the technical limitations of prime editing will take time to resolve. Maybe this bottleneck would not be a significant issue if in that population of transduced cells, basal cells were effectively edited. This would lead to a repopulation of airway epithelial cells with edited alleles and rescued CFTR function, thereby amplifying the therapeutic impact of low-efficiency prime editing. Important for this study, what cell types in the differentiated airway epithelia were transduced by HDAd? Are basal cells included in this population?

2) Why was functional rescue of p.W1282X-CFTR prime edited differentiated epithelial cells not reported? It would seem that even with as low as 1.5% editing there would be measurable rescue of CFTR function by Ussing chamber assays for measurement of short circuit current (Isc). Indeed, the CFTR functional rescue is often significantly higher than the genome editing efficiency.

Reviewer #2: I have reviewed the manuscript numbered PONE-D-23-20582, titled “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, submitted by Chao L., Zhong L., et al.

This article, presenting an original research, is very interesting and very clearly described.

In the manuscript, the authors have developed a novel PRIME editing system for the correction of the W1282X CFTR mutation.

The authors did a very good initial screening job for the components of the PRIME editing System (as reported also in supplementary information) and then, validated the best one in iPSCs generated from nasal epithelial cells of a homozygous W1282X cystic fibrosis patient.

PE-iPSCs were also differentiated into airway epithelial cells and lung organoids, for testing the restored CFTR function. As a very important point to become a therapeutic modality, the Authors tested the PRIME editing system directly in differentiated airway epithelial cells, using an HDAd vector containing all the sequences needed for the prime editing system (all-in –one vector).

In conclusion, the Authors demonstrated the correction of the point mutation albeit in a limited number (2.5-5%) of iPS-derived epithelial cells and, very importantly, getting no indels effect though the DNA.

These results could be very useful for the development of an in-vivo PRIME editing system for stop mutation in CF.

The only suggestion to the Authors could be to add a sentence in the discussion, in particular in the paragraph stating the editing efficiency, indicating (if it is known) what could hypothetically be the correction rate to be reached by PRIME-CRISPR/Cas9-based systems, in order to get a therapeutic level of CFTR for CF patients.

Other specific comments:

-Are Results and Conclusions presented in an appropriate fashion and are supported by the data?

YES. Results are very well written, linear, and complete. The same is for the Conclusions (see my only suggestion above).

-Is the article presented in an intelligible fashion and written in standard English?

YES

- Does the research meet all applicable standards for the ethics of experimentation and research integrity?

YES

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2023 Nov 29;18(11):e0295009. doi: 10.1371/journal.pone.0295009.r002

Author response to Decision Letter 0

18 Oct 2023

We sincerely thank the editor’s comments and feedback. We have updated and formatted the revised manuscript accordingly. Below are our point-to-point responses.

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

� We formatted the revised manuscript according to the style requirements. This includes changing level 1 and 2 headings to appropriate fonts, abiding by the requirements for figure citations/captions, and updating the style of supporting information citations and acknowledgments. We also updated the title, author, and affiliation style to abide by the guidelines.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

"R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594."

We note that you have provided funding information that is not currently declared in your Funding Statement.

� We have updated the funding information in the revised submission.

However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"R.Z. is supported by Cystic Fibrosis Foundation Research Grant ZHAO19G0, UAB Cystic Fibrosis Research Center Pilot Grant (ROWE15R0), and NIH R01OD026594.

� Funding information has been deleted from the Acknowledgements section.

NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

� The below statement has been included in the cover letter.

“The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

3. Thank you for stating the following in the Competing Interests section:

"I have read the journal's policy and the authors of this manuscript have the following competing interests:

S.M.R. provided consulting services and received grants from Novartis, TranslateBio, Galapagos/Abbvie, Synedgen/Synspira, Eloxx, Vertex Pharmaceuticals, Ionis, Astra Zenica, Renovion, Cystetic Medicines, and Arcturus. S.M.R. is the inventor or co-inventor of several patents and held stock or stock options of Synedgen/Synspira and Renovion. "

Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

� The below statement has been included in the cover letter and revised manuscript.

“This does not alter our adherence to PLOS ONE policies on sharing data and materials. "

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

� “data not shown” has been removed from the revised manuscript because it is not a core part of the reported study.

5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

� Captions for Supporting Information have been added to the end of the revised manuscript to meet the guidelines.

[Note: HTML markup is below. Please do not edit.] 

We sincerely thank the reviewers for their positive comments and critical feedback, which we have used to revise and strengthen our manuscript. Please find the below point-to-point responses. We anticipate that our study on the correction of CFTR W1282X mutation by prime editing will be of great interest to readers of PLOS ONE.

Reviewer #1: The study “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, by Li and colleagues is of high quality and of interest to a broad audience. The results presented in the main figures of the manuscript provide a concise and impactful study on the use of prime editing to “fix” CFTR mutation p.W1282X. The major advances presented here are the use of an “all-in-one” helper dependent Adenovirus (HDAd) to efficiently deliver all of the prime editing machinery to iPSC-derived airway epithelial cells and restore p.W1282X-CFTR function. Because of the size of the payload, this has previously not been able to be achieved. This reviewer found much of the interesting biology to be in the supplemental figures, where a good amount of optimization was performed. There are only a few comments to address.

Comments:

1) The authors touted the ability of delivering all of the machinery in a helper dependent adenovirus (HDAd) system for efficient editing. However, even with a plasmid system containing all of the PE machinery and high transfection efficiency, the editing efficiency was only 0.36% in iPSCs. (Fig 1D). With the HDAd system providing ~70% transduction efficiency of differentiated airway epithelial cells only corrected 2-3% of the p.W1282X alleles. Indeed, the bottleneck for correction of p.W1282X CF mutations is the overall efficiency of prime editing, not delivery. While there is nice discussion on this issue, the technical limitations of prime editing will take time to resolve. Maybe this bottleneck would not be a significant issue if in that population of transduced cells, basal cells were effectively edited. This would lead to a repopulation of airway epithelial cells with edited alleles and rescued CFTR function, thereby amplifying the therapeutic impact of low-efficiency prime editing. Important for this study, what cell types in the differentiated airway epithelia were transduced by HDAd? Are basal cells included in this population?

� We agree that the observed low correction efficiency could hold therapeutic significance if the corrected cells are predominantly basal cells. For iPSC differentiation into airway epithelium, we primarily followed the published protocol (ref. 29 and 30), which demonstrated the presence of basal cells in iPSC-derived epithelium. HDAd can effectively transduce airway basal cells in animal models (ref. 22). However, determining prime editing-corrected cell types is technically challenging. Our current experimental system detects the prime editing-mediated single nucleotide substitution solely by ddPCR. The ddPCR procedure prevents simultaneous cell type identification.

A potential solution to this important question is to construct a W1282X reporter iPSC line, in which a GFP reporter will be fused in-frame to the 3’ end of the mutant CFTR W1282X open reading frame. Because of the W1282X mutation, uncorrected airway cells would remain GFP negative but airway cells with correction would become GFP positive. The GFP-positive cells can then be FACS-sorted for marker staining and single-cell sequencing analysis. However, this approach involves a significant amount of efforts and we believe it should become a follow-up study of the current report. We agree this is an important point that should be further explored. We have included a discussion on the potential implications of correcting basal cells in the revised manuscript (Page 15).

2) Why was functional rescue of p.W1282X-CFTR prime edited differentiated epithelial cells not reported? It would seem that even with as low as 1.5% editing there would be measurable rescue of CFTR function by Ussing chamber assays for measurement of short circuit current (Isc). Indeed, the CFTR functional rescue is often significantly higher than the genome editing efficiency.

� We agree with the reviewer and have conducted the recommended experiment (supplemental Figure S4). In the prime-edited airway epithelium, we detected a minor Isc spike following FSK stimulation, a response absent in the unedited controls. When compared to the PE-iPSC-derived airway epithelium (Fig. 3B), the amplitude of the FSK-induced Isc is approximately 20-fold lower, which is in line with the expected functional CFTR allele frequencies (~2.5% vs. 50%). However, we did not observe a VX-770-induced Isc change, which could be attributed to the generally low Isc signals of the HDAd-corrected epithelium or other unidentified factors. We have included these new data in the revised manuscript (Fig. S4 and page 13).

Reviewer #2: I have reviewed the manuscript numbered PONE-D-23-20582, titled “Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells”, submitted by Chao L., Zhong L., et al.

This article, presenting an original research, is very interesting and very clearly described.

In the manuscript, the authors have developed a novel PRIME editing system for the correction of the W1282X CFTR mutation. The authors did a very good initial screening job for the components of the PRIME editing System (as reported also in supplementary information) and then, validated the best one in iPSCs generated from nasal epithelial cells of a homozygous W1282X cystic fibrosis patient. PE-iPSCs were also differentiated into airway epithelial cells and lung organoids, for testing the restored CFTR function. As a very important point to become a therapeutic modality, the Authors tested the PRIME editing system directly in differentiated airway epithelial cells, using an HDAd vector containing all the sequences needed for the prime editing system (all-in –one vector).

In conclusion, the Authors demonstrated the correction of the point mutation albeit in a limited number (2.5-5%) of iPS-derived epithelial cells and, very importantly, getting no indels effect though the DNA.

These results could be very useful for the development of an in-vivo PRIME editing system for stop mutation in CF.

The only suggestion to the Authors could be to add a sentence in the discussion, in particular in the paragraph stating the editing efficiency, indicating (if it is known) what could hypothetically be the correction rate to be reached by PRIME-CRISPR/Cas9-based systems, in order to get a therapeutic level of CFTR for CF patients.

� We agree that this is an important point. It is believed that restoration of 10 to 35% of CFTR activities would be required to mitigate pulmonary morbidity (PMID: 15510065). Based on this estimation, a correction level ranging from 10 to 35% mutant alleles would be necessary. However, if the corrected cells primarily comprise basal cells, therapeutic advantages could potentially be attained with lower editing efficiency. We have included this discussion in the revised manuscript (page 15).

Attachment

Submitted filename: Rebuttal_03.pdf

Click here for additional data file. (213KB, pdf)

Decision Letter 1

Alessio Branchini

14 Nov 2023

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

PONE-D-23-20582R1

Dear Dr. Zhao,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Alessio Branchini

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: John D. Lueck

**********

Acceptance letter

Alessio Branchini

17 Nov 2023

PONE-D-23-20582R1

Prime editing-mediated correction of the CFTR W1282X mutation in iPSCs and derived airway epithelial cells

Dear Dr. Zhao:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Alessio Branchini

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Appendix. It includes four supporting figures, figure legends, and a supporting table.

    (DOCX)

    Click here for additional data file. (2.6MB, docx)
    S2 Appendix. W1282X organoid FIS assay.

    (WMV)

    Click here for additional data file. (5.5MB, wmv)
    S3 Appendix. PE organoid FIS assay.

    (WMV)

    Click here for additional data file. (5.4MB, wmv)
    Attachment

    Submitted filename: Rebuttal_03.pdf

    Click here for additional data file. (213KB, pdf)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

    Articles from PLOS ONE are provided here courtesy of PLOS

    RESOURCES