Study approval and human subjects
The study was approved by the responsible local ethics committees (Institutional Review Board of the University Medical Center Utrecht and University Medical Center Groningen (STEM: 10-402/K; TcBio 14-008; Metabolic Biobank: 19-489)). For cystic fibrosis organoids, collection of patient tissue and data was performed following the guidelines of the European Network of Research Ethics Committees (EUREC). Tissue biopsies from the liver of a patient with ABCB4 deficiency (PFIC3) were obtained during a liver transplant procedure in the UMCG, Groningen. Rectal biopsies used for intestinal organoid culture from a patient with ATP8B1 deficiency (PFIC1) and skin biopsies used for fibroblasts culture from a patient with homozygous POLGA467T mutations were obtained at the outpatient clinic in the UMCU, Utrecht. Biobanked intestinal organoids are stored and cataloged (https://huborganoids.nl/) at the foundation Hubrecht Organoid Technology (http://hub4organoids.eu). All biopsies were used after written informed consent.
ClinVar database computational analysis
Information for all pathogenic mutations shorter than 51 bp was obtained from the ClinVar database29, accessed October 2020. Genomic sequences flanking these mutations were obtained from RefSeq30 accessed October 2020, using the SPDI data model31 and a custom python script. The −10 to +4 bp region around the target sites were searched for NGG, NAN, or NGN PAMs. The efficiency of prime editing using NGG PAMs was predicted using PE_Position and PE_type random forest models, provided by Kim et al.12. Figures were made in python using Matplotlib32. The code used for this analysis is available on GitHub (https://github.com/JBaijens/PE_prediction).
Plasmid cloning
FluoPEER plasmids were cloned using the backbone of the pmGFP-P2A-K0-P2A-RFP (Addgene #105686) stalling reporter (SR) plasmid, which was a gift from Ramanujan Hegde. This plasmid was cut directly upstream of the K0-SR domain with SalI and Acc65I for 16 hours at 37 °C, creating ‘TCGA’ and ‘GTAC’ overhangs, after which the 6 kb fragment was isolated from gel. Genomic insert oligos containing 5′ ‘TCGACC’ and 3′ ‘G’ overhangs on the top oligo, and 5′ ‘GTACC’ and 3′ ‘GG’ overhangs on the bottom oligo were annealed and inserted using a conventional ligation protocol (see Supplementary Note 1). Note that a finalized fluoPEER plasmid still contains the K0-SR domain, which is not shown in the schematic representations in Figs. 1a, d, e and 2a, f as it is not important for the working mechanism of fluoPEER. Cloning of pegRNA and epegRNA plasmids was performed according to previously described protocols1,4. In brief, the pU6-pegRNA-GG-Vector (Addgene #132777) or the pU6-tevopreq1-GG-acceptor (Addgene #174038) was digested for 16 h with BsaI-HFv2 (NEB), after which the 2.2 kb fragment was isolated from gel. Oligonucleotide duplexes of the pegRNA spacer, pegRNA extension, and pegRNA scaffold sequences were ordered containing the appropriate overhangs and subsequently annealed. The annealed pegRNA duplexes were ligated into the pU6-pegRNA-GG-Vector using Golden Gate assembly with BsaI-HFv2 (NEB) and T4 DNA ligase (NEB) in a protocol of 12 cycles of 5 min at 16 °C and 5 min at 37 °C. For cloning of sgRNAs used for PE3, we replaced the BsmBI restriction sites of the BPK1520 plasmid with BbsI restriction sites using PCR, which allowed direct ligation of sgRNA-spacer duplexes33. All fluoPEER insert, pegRNA, and sgRNA sequences used in this work are listed in Supplementary Data file 2 and were synthesized by Integrated DNA Technologies (IDT). pCMV-PE2 (Addgene #132775), pU6-pegRNA-GG-acceptor (Addgene #132777), and pU6-tevopreq1-GG-acceptor (Addgene #174038) were gifts from David Liu; BPK1520 (Addgene #65777) was a gift from Keith Joung.
Cloning of flexible PE2s, flexible PE2*, and SpRY-PE4max
Using PCR and In-Fusion cloning (Takara Bio), the NGG PAM-recognition domain of the prime editor protein (PE2) was replaced with the corresponding domains in NG-ABEmax34, SpG-ABEmax, or SpRY-ABEmax8, to create NG-, SpG-, and SpRY-PE2, respectively. NG-ABEmax was a gift from David Liu (Addgene #124163). SpG- and SpRY-ABEmax were gifts from Benjamin Kleinstiver (Addgene plasmids #140002 and #140003). PE2* variants with improved nuclear localization sequences (NLSs) were adapted from the NGG-PE2* developed by Liu et al.3 and were cloned by PCR and In-Fusion cloning (Takara Bio). Successful cloning of all plasmids was confirmed by Sanger sequencing. Using PCR and In-Fusion cloning (Takara Bio), SpRY-PE4max was created by replacing the PAM-recognition domain of PE4max5 (#174828) with the corresponding domain of SpRY-ABEmax (#140003)8. pCMV-PEmax-P2A-hMLH1dn (PE4max, Addgene #174828) was a gift from David Liu.
Organoid culture
Liver and intestinal organoids were grown under standard culture conditions according to previously described protocols35,36. In short, liver organoids were plated in matrigel (Corning) and maintained in human liver expansion medium (hL-EM), consisting of AdDMEM/F12 (Gibco) supplemented with, GlutaMAX (1x, Gibco), HEPES (1x, Gibco), PenStrep (1x, Gibco), 2% B27 without vitamin A (Gibco), 1.25 mM N-Acetylcysteine (Sigma), 10 mM Nicotinamide (Sigma), 10 nM gastrin (Sigma), 10% RSPO1 conditioned media (homemade), 50 ng/ml EGF (Peprotech), 100 ng/ml FGF10 (Peprotech), 25 ng/ml HGF (Peprotech), 5 mM A83-01 (Tocris), and 10 mM FSK (Tocris). Small intestine and colon organoids were plated in matrigel and maintained in human small intestine expansion medium (hSI-EM), consisting of AdDMEM/F12 (Gibco) supplemented with, GlutaMAX (1x, Gibco), HEPES (1x, Gibco), PenStrep (1x, Gibco), 50% WNT3A-, 20% RSPO1-, and 10% NOG(gin)-conditioned medium (all homemade), 2% B27 with vitamin A (Gibco), 1.25 mM N-Acetylcysteine, 10 mM Nicotinamide, 50 ng/ml murine-EGF (Peprotech), 500 nM A83-01, and 10 mM SB202190 (Sigma). The medium was changed every 2–4 days and organoids were passaged 1:4–1:8 each week. After thawing, organoids were passaged at least once before electroporation.
Cell culture, lentiviral production, and lentiviral transduction
HEK293T, Caco-2 and HeLa cells were obtained from the ATCC.
Fibroblasts were maintained and split every 7 days in standard medium, consisting of F-12 Nut Mix (Ham) (Gibco), 10% FBS (Gibco), and PenStrep (1x, Gibco). HEK293T, Caco-2, and HeLa cells were maintained and split every 4-5 days in standard medium, consisting of DMEM + GlutaMAX (1x, Gibco), 10% FBS (Gibco), and PenStrep (1x, Gibco). For production of lentivirus, HEK293T cells were plated in a 145 mm CELLSTAR dish (Corning) in standard medium without PenStrep and transfected 24 hours later (at 50–60% confluence) with a mix of 10 μg of the pLenti-CMV-GFP-Hygro plasmid, 5 μg of psPAX2, 5 μg of pMD2.G, and 60 μl of polyethylenimine (1 mg/ml). pLenti-CMV-GFP-Hygro was a gift from Eric Campeau & Paul Kaufman (Addgene #17446). psPAX2 and pMD2.G were gifts from Didier Trono (Addgene plasmids #12260 and #12259). 24 h after transfection, the medium was replaced with standard medium with PenStrep, and virus-containing medium was harvested at 48 and 96 hours after transfection. Medium was centrifuged at 400 × g for 5 min and supernatants were filtered through a 0.22-μm filter, after which virus particles were concentrated by ultracentrifugation at 50,000 × g for 2 hours and resuspension in 1 ml DMEM. HEK293T cells were transduced at an MOI of 2 for 24 h in the presence of polybrene (8 μg/μl) and analyzed by FACS 14 days after transduction to confirm stable GFP expression.
Transfection of HEK293T cells
HEK293T cells were plated in standard medium at a density of 50,000 cells per well in a 96-well plate 1 day prior to transfection. HEK293T cells were transfected with 0.1 µg fluoPEER plasmid, 0.25 µg prime editor plasmid, 0.1 µg pegRNA plasmid, and optionally 0.05 µg nicking-gRNA plasmid in a mix of 25 µl OptiMEM and 0.5 µl lipofectamine 2000 for each well.
Electroporation of organoid cells
Before electroporation, organoids were grown under standard culture conditions. Four wells containing organoids were then dissociated for each condition using TrypLE (Gibco) for 4–5 min at 37 °C, after which mechanical disruption was applied through pipetting. Cells were washed once using Advanced DMEM/F12, resuspended in 80 µl OptiMEM containing Y-27632 (10 µM), and 20 µl DNA mixture was added. For prime editing, the DNA mixture contained 4 µg fluoPEER, 12 µg prime editor plasmid, 4 µg pegRNA plasmid, and 2 µg nicking sgRNA plasmid. The cell-DNA mixture was transferred to an electroporation cuvette and electroporated using a NEPA21 electroporator (NEPA GENE) with 2× poring pulse (voltage: 175 V, length: 5 ms, interval: 50 ms, polarity: +) and 5× transfer pulse (voltage: 20 V, length: 50 ms, interval: 50 ms, polarity ± ), as previously described37. Cells were removed from the cuvette and transferred into 500 µl OptiMEM containing Y-27632 (10 µM). After 20 minutes, cells were plated in 120 µl matrigel divided over four wells. Upon polymerization of the Matrigel, hl-EM or hSI-EM was added containing Y-27632 (10 µM).
Transfection of fibroblast and HeLa cells
Skin-derived fibroblasts were grown under standard culture conditions and plated on 12-well plates 3 days prior to transfection so that confluency was 60–70% at transfection. HeLa cells were grown under standard culture conditions and plated on 24-well plates 1 day prior to transfection so that confluency was 60–70% at transfection. For prime editing experiments, fibroblasts and HeLa cells were transfected with 0.12 µg fluoPEER plasmid, 0.19 µg prime editor plasmid, 0.05 µg pegRNA plasmid and 0.05 µg nicking-gRNA plasmid in a mix of 8.4 µl OptiMEM, 0.6 µl lipofectamine 3000, and 0.4 µl P3000 reagent. For base editing experiments, the DNA mix consisted of 0.14 µg fluoPEER plasmid, 0.2 µg ABE8e-TadA (V106W) plasmid, and 0.06 µg sgRNA. ABE8e (TadA-8e V106W) was a gift from David Liu (Addgene #138495).
FACS
Organoids and cell lines were harvested and dissociated to single cells using TrypLE (Gibco) or Trypsin (Gibco), respectively, after which cells were resuspended in FACS buffer (phosphate-buffered saline with 2 mM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin). Prior to FACS, cells were filtered through a 5 ml Falcon polystyrene test tube (Corning). Flow cytometry was performed on the FACS Fortessa (BD) and sorting was performed on the FACS FUSION (BD) using FACS Diva software (BD). Sorted cells were collected in culture medium and spun down. Gating strategy for cells included for fluoPEER analysis is shown in Supplementary Fig. 12a. The ratio was calculated by dividing the average measured Cherry signal by the average measured GFP signal for all GFP+ cells.
Genotyping
Sorted cells were harvested using the Quick-DNA microprep kit (Zymogen) according to manufacturer’s protocols. PCR was performed on the genomic region of interest using the Phusion polymerase (ThermoFisher) or Q5 polymerase (NEB) and purified using the QIAquick PCR Purification Kit (Qiagen) according to manufacturer instructions. The PCR product was sent for Sanger sequencing to EZSeq Macrogen Europe. All PCR and sequencing primers used are listed in Supplementary Data file 2.
High-throughput DNA sequencing of genomic DNA samples
Genomic sites of interest were amplified from genomic DNA samples and sequenced on an Illumina iSeq 100 as previously described38. In short, PCR primers containing Illumina forward and reverse adapters (Supplementary Data file 2) were used in a first amplification reaction (PCR1) of 25 µl using Q5 polymerase (NEB) to amplify the genomic region of interest. In a second round of PCR (PCR2, 25 µl), 1 µl of each PCR1 was barcoded with unique Truseq DNA Index primers (Illumina) and isolated from gel. DNA concentration was measured by fluorometric quantification (Qubit, ThermoFisher Scientific) and sequenced on an Illumina iSeq 100 instrument according to the manufacturer’s protocols to create 2 × 150 bp paired-end reads. The resulting FASTQ files were analyzed with the RGEN PE-analyzer, using the unedited sequence as the reference sequence and the prime-edited sequence as the intended sequence39. Prime editing efficiency was calculated as the percentage of (RGEN PE-reads/RGEN more than minimum frequency reads). For unwanted byproduct analysis at the pegRNA or nickase sgRNA site, a comparison range (R) of 30 bp or 70 bp was used so that 60 bp or 140 bp flanking the predicted nicking site were considered. Frequency of indels was calculated as the percentage of (RGEN reads with unwanted inserts and deletions/RGEN more than minimum frequency reads).
RNA sequencing
HEK293T cells were transfected with fluoPEER, PE2, pegRNA, and nicking sgRNA plasmids and FACS sorted after 48 h. Total RNA was isolated using Trizol LS reagent (Invitrogen) and stored at –80 °C until further processing. mRNA was isolated using Poly(A) Beads (NEXTflex). Sequencing libraries were prepared using the Rapid Directional RNA-Seq Kit (NEXTflex) and sequenced on a NextSeq500 (Illumina) to produce 75 base long reads (Utrecht DNA Sequencing Facility). Sequencing reads were mapped against the reference genome (hg19 assembly, NCBI37) using BWA40 package (mem –t 7 –c 100 –M –R). Raw reads were further analyzed as described under ‘Data analysis’.
Chemical cell cycle synchronization
Chemical cell cycle synchronization using thymidine and nocodazole was performed as described previously41,42. In short, 50,000 HEK293T cells or 10,000 Caco-2 cells were plated in 24-well plates. After 8 h, 2 mM thymidine was added to the cells. 17 h later, cells were washed twice and medium was replaced with standard culture medium. 8 h later, 2 mM thymidine was readded to the cells. At the same time point, 200 ng/ml nocodazole was added for the nocodazole treatment. 20 h later, cells were washed twice, medium was replaced with standard culture medium, and prime editing was performed by transfection of 0.25 µg prime editor plasmid and 0.1 µg pegRNA plasmid in a mix of 100 µl OptiMEM and 0.3 µl lipofectamine 2000 for each well. 48 h later, transfected cells were sorted using flow cytometry and genotyping was performed as described above.
fluoPEER cell cycle analysis
200 ng/ml nocodazole was added to HEK293T cells 20 h before FACS analysis as a control for cells in G2 phase. 24 h before fluoPEER read-out and genomic Sanger sequencing, HEK293T cells were transfected. 60 min before harvesting for FACS analysis, 10 µg/ml Hoechst 33342 (ThermoFisher) was added to the culture medium. Gating strategy for G1, S, and G2 phases of the cell cycle is shown in Supplementary Fig. 12b.
Data analysis
Flow cytometry data were analyzed using FlowJoTM Software. RNA sequencing was analyzed using DESeq2 in RStudio43, gene set enrichment analysis44, and enrichR45. All figures were made in Prism (GraphPad Software) or GGPlot246 in RStudio. Sanger sequencing was quantified using EditR47 or Tide48. Sanger sequencing chromatograms were made in Benchling. NGS data were quantified and analyzed using RGEN PE-analyzer39.
Statistics and reproducibility
No pre-specified effect size was calculated, and no statistical method was used to predetermine sample size. For comparisons of multiple groups, an ordinary one-way ANOVA with Holm–Sidak correction for multiple comparisons was used and performed in Prism (GraphPad Software). Statistical tests were appropriate for comparisons being made; assessment of variation was carried out but not included. Experiments were not randomized. Reproducibility: Fig. 1c representative of three biologically independent replicates from one experiment. In Fig. 1d, each point in the dot-plot represents the mean of three (reporter rank) or two (genomic rank) replicates in two independent experiments. Each point in graphs of Fig. 1e represents the mean of at least two biologically independent replicates for each prime editing condition in a single experiment. Figure 1f representative of (1) 14/20 clonally picked intestinal organoids from two different patients with biallelic CFTRF508del mutations, that showed swelling after addition of 1 µM forskolin to the medium, (2) 2/10 clonally picked liver-derived organoids from a patient with biallelic ABCB4E1012X mutations, (3) 3/10 clonally picked intestinal organoids from a patient with biallelic ATP8B1R600Q mutations, and (4) 2/10 clonally picked liver-derived organoids from a healthy control, in which biallelic IARS1I1174N were created; 4/10 clonally picked organoids from the same experiment showed monoallelic IARS1I1174N mutations.
Figure 2a is representative of two replicates in two independent experiments. In Fig. 2b each dot represents the ratio of two bars (PE2* vs. PE2) in Supplementary Fig. 8a, b. In Supplementary Figure 8a, b, each bar represents the mean of 2–3 biologically independent replicates. Figure 2c, d, e are representative of three biologically independent replicates from one experiment. Figure 2f shows representative data from one experiment. Figure 3a, b are representative of at least two biologically independent replicates from one experiment. Data from Fig. 3c is based on one transfection, of which 8 conditions of 100 GFP + cells and 2 conditions of 100 GFP + RFP + cells were sorted. Figure 3d is representative of three biologically independent replicates from one experiment. Figure 3e is representative of two or three biologically independent replicates from one experiment. Figure 4b–d are data from RNA sequencing of two biologically independent replicates for both conditions (GFP + RFP-/GFP + RFP + ) for each edit (CTNNB1/IARS1) from one experiment. In Fig. 4e HEK293T, each dot represents the mean of three biologically independent replicates, three independent experiments were performed. In Fig. 4e Caco-2, each dot represents a biologically independent replicate from one experiment.
Supplementary Figure 4 is representative of two replicates from two independent experiments. The fluoPEER data in Supplementary Fig. 5a, b are representative of three biologically independent replicates in one experiment; fluoPEER data in supplementary Fig. 5c is representative of two biologically independent replicates in one experiment that was characteristic of two repeated experiments. Supplementary Fig. 6a–c are representative of two biologically independent replicates per condition in one experiment; for representation of the percentage of GFP+ cells, data from different conditions were pooled. Supplementary Fig. 6d is based on data from three biologically independent replicates of sorted cells to seed 100% GFP + conditions. Each pegRNA-PE2-fluoPEER combination from Supplementary Fig. 7 was tested in at least two independent transfection experiments. Supplementary Figure 7 is representative of two or three biologically independent replicates from one characteristic experiment. Supplementary Figure 7b, c representative of clonally picked liver organoids with monoallelic (two clones) or biallelic (two clones) IARS1I1174N mutations. Supplementary Fig. 7d representative of 20 clonally picked liver-derived organoids from a patient with biallelic MUTR329H mutations. Supplementary Figure 8c is representative of three biologically independent replicates from one experiment. Supplementary Figure 9 shows FACS plots of single conditions that are representative of two independent transfection experiments. Supplementary Figure 10a–d represent data from two biologically independent replicates for each experimental group. Supplementary Figure 10e represents data from three biologically independent replicates for two independent biological replicates. Supplementary Figure 11 data is representative of three biologically independent replicates from NGS. Supplementary Figure 12 is representative FACS data for all FACS experiments shown.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
