Protocol for generating multiplexed prime-edited monoclonal cell lines in porcine fetal fibroblasts

Summary

Porcine fetal fibroblasts (PFFs) serve as standard donor cells for generating cloned pigs, and prime editing (PE) enables precise genome modification. Here, we describe a protocol for generating multiplexed prime-edited monoclonal cell lines in PFFs. We describe steps for pegRNA/ngRNA design and screening, plasmid electroporation, nocodazole treatment, puromycin selection, and monoclonal isolation and genotyping. Although demonstrated by introducing three Alzheimer’s disease-associated pathogenic mutations, the pipeline can be readily adapted to multiplex PE of other endogenous loci in porcine cells.

For complete details on the use and execution of this protocol, please refer to Liu et al.¹

Graphical abstract

Highlights

• •Instructions for multiplex prime editing using pvPE, nocodazole, and puromycin in PFFs
• •Guidance on selecting functional pegRNA/ngRNA pairs before clone isolation
• •Steps for generating and validating prime-edited monoclonal cell lines

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Porcine fetal fibroblasts (PFFs) serve as standard donor cells for generating cloned pigs, and prime editing (PE) enables precise genome modification. Here, we describe a protocol for generating multiplexed prime-edited monoclonal cell lines in PFFs. We describe steps for pegRNA/ngRNA design and screening, plasmid electroporation, nocodazole treatment, puromycin selection, and monoclonal isolation and genotyping. Although demonstrated by introducing three Alzheimer’s disease-associated pathogenic mutations, the pipeline can be readily adapted to multiplex PE of other endogenous loci in porcine cells.

Before you begin

Timing: 3–6 days (step 1)

Timing:4–6h (step 2)

This protocol describes multiplexed prime editing of endogenous genes in porcine fetal fibroblasts (PFFs). Because the activity of prime editing guide RNAs (pegRNAs) and nicking guide RNAs (ngRNAs) can vary substantially depending on the target loci and sequence context, this workflow includes an initial phase for the design and evaluation of multiple candidate pegRNA/ngRNA combinations. Optimal pairs are subsequently selected for multiplexed prime editing, followed by single-cell cloning and genotyping.

In this protocol, prime editing is performed by co-electroporation of a porcine endogenous retrovirus-derived prime editor (pvPE) plasmid together with pegRNA/ngRNA expression constructs into PFFs. The pvPE system was developed in our previous study, in which pvPE configurations showed higher intended editing efficiency and reduced editing byproducts compared with matched MMLV-RT-based prime editors. Nocodazole treatment was also shown to increase prime editing efficiency.¹ Therefore, transient nocodazole treatment is applied as an efficiency-enhancing step in this workflow. Puromycin selection is subsequently used as a common enrichment strategy in PFFs to increase the proportion of transfected cells prior to downstream editing efficiency analyses and monoclonal cell line derivation.

Before starting, ensure that early-passage PFFs and the required cloning vectors for pegRNA and ngRNA construction are available.

• 1.Preparation of early-passage PFFs.

Note: PFFs used in this protocol were isolated from embryonic day 30 porcine fetuses¹ and cryopreserved before use. Cryopreserved PFFs should be thawed, expanded, and allowed to recover for at least one passage prior to electroporation.

CRITICAL: Use actively proliferating, early-passage PFFs exhibiting typical fibroblast-like morphology. Avoid over-confluent, senescent, or slow-growing cultures, as these cells often show reduced survival following puromycin selection and poor monoclonal outgrowth.

• 2.
  Preparation of linearized pegRNA and ngRNA backbone vectors

  • a.
    Digest the pU6-pegRNA-GG and pGL3-U6-sgRNA-EGFP backbone plasmids with BsaI at 37°C for 3 h, followed by heat inactivation at 80°C for 20 min.

    Note: The pU6-pegRNA-GG and pGL3-U6-sgRNA-EGFP backbone plasmids serve as cloning backbones for pegRNAs and ngRNAs, respectively. Both vectors are linearized by BsaI digestion before ligation of the corresponding annealed oligonucleotides.

  • b.
    Separate the digestion products by agarose gel electrophoresis. Excise the desired linearized backbone fragments from the gel and purify them using a commercial gel extraction kit.

    Note: The expected digestion fragments are approximately 2.2 kb for pU6-pegRNA-GG and 5 kb for pGL3-U6-sgRNA-EGFP. Quantify the purified DNA using a spectrophotometric or fluorometric assay and store it at −20°C until use.

    CRITICAL: Complete digestion of the backbone vectors is essential. Incomplete digestion or carryover of undigested plasmid can substantially reduce cloning efficiency.

Innovation

To our knowledge, this protocol represents the first integrated workflow for generating multiplexed prime-edited monoclonal cell lines in PFFs. Existing prime-editing protocols primarily focus on pegRNA design, editor delivery, and bulk editing-efficiency evaluation. In contrast, the present protocol not only provides a strategy for evaluating candidate pegRNA/ngRNA combinations in PFFs, but also establishes a practical framework for efficient prime editing and monoclonal cell line generation in this cell type. Specifically, the workflow includes co-electroporation of prime-editing components for simultaneous multi-locus editing, nocodazole-mediated enhancement, puromycin-based enrichment, limiting-dilution cloning, manual colony isolation, monoclonal expansion, and clone-level genotyping.

By evaluating pegRNA/ngRNA activity before monoclonal cell line derivation, the workflow reduces the time and effort required for expansion and genotyping of non-productive clones. The described strategy provides a practical and scalable framework for multiplexed editing of endogenous loci in porcine cells, with clearly defined steps that support reproducible implementation across independent experiments.

Institutional permissions

All experimental procedures were approved by the Ethics Committee of Jiangxi Agricultural University under protocol number JXAULL-2024-01-016.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Bacterial and virus strains
E. coli DH5α competent cells	Tsingke Biotech	Cat# DLC101
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco	Cat# 11995-073
1× Phosphate-buffered saline (PBS), pH 7.4	Gibco	Cat# 10010049
0.25% trypsin-EDTA solution	Sigma-Aldrich	Cat# T4049
BsaI-HFv2	NEB	Cat# R3733S
T4 DNA ligase	NEB	Cat# M0202S
Fetal bovine serum (FBS)	Excell Bio	Cat# FSD500
Penicillin-streptomycin	Gibco	Cat# 15070063
Cell lysis buffer II	Gibco	Cat# FNN0021
Proteinase K	Solarbio	Cat# P9460
PrimeSTAR® Max DNA Polymerase	Takara	Cat# R045B
Vaseline	Sangon Biotech	Cat# 8009-03-8
Entranster-E	Engreen	Cat# 98668-20
Nocodazole	MedChemExpress	Cat# HY-13520
Puromycin	Sigma-Aldrich	Cat# P9620
Critical commercial assays
TaKaRa MidiBEST Endo-free Plasmid Purification Kit	Takara	Cat# 9783
Deposited data
Raw data	Liu et al.1	N/A
Experimental models: Cell lines
PFFs	Liu et al.1	N/A
Recombinant DNA
pU6-pegRNA-GG-acceptor	Addgene	Cat# 132777
pGL3-U6-sgRNA-EGFP	Addgene	Cat# 107721
pvPE-V3-Puro	Addgene	Cat# 240289
Software and algorithms
Prism 9.5	GraphPad	graphpad.com
pegFinder	Chow et al.2	http://pegfinder.sidichenlab.org/
PrimeDesign	Hsu et al.3	https://github.com/pinellolab/PrimeDesign
PRIDICT 2.0	Mathis et al.4	https://www.pridict.it/
CRISPResso2	Clement et al.5	https://crispresso.pinellolab.org/submission
SnapGene	Dotmatics Software	https://www.snapgene.com/
Other
10-cm culture dish	Thermo Fisher	Cat# 150466
6-cm cell culture dish	Thermo Fisher	Cat# 150288
6-well cell culture plate	Thermo Fisher	Cat# 140675
24-well cell culture plate	Thermo Fisher	Cat# 142475
1.5 mL sterile centrifuge tube	Axygen	Cat# MCT-150-C-S
10 mL sterile serum pipette	Labselect	Cat# SP-013-10
0.2 mL 8-strip tube	Labselect	Cat# PST-0208-AFT-C
4D-Nucleofector® Core Unit	Lonza	Cat# AAF-1003B
4D-Nucleofector® X Unit	Lonza	Cat# AAF-1003X
P3 Primary Cell 4D-Nucleofector® X Kit S	Lonza	Cat# V4XP-3032
P3 Primary Cell 4D-Nucleofector® X Kit L	Lonza	Cat# V4XP-3024

Materials and equipment

Prepare regular growth medium and clonal growth medium using the following formulations.

Growth media formulations

Reagent	Regular growth medium	Clonal growth medium
DMEM	500 mL	500 mL
FBS	60 mL	90 mL
Penicillin-streptomycin	5.6 mL	6 mL
Total	565.6 mL	596 mL

Note: The clonal growth medium contains a higher serum concentration and is used during monoclonal cell line establishment to support efficient clonal formation and outgrowth. The medium can be stored at 4°C for up to one month.

Cell lysis solution

Reagent	Final concentration	Amount
Cell lysis buffer II	45% (v/v)	450 μL
10 mg/mL proteinase K solution	600 μg/mL	60 μL
ddH2O	49% (v/v)	490 μL
Total	N/A	1 mL

Note: Dissolve proteinase K in an appropriate volume of distilled water to prepare a 10 mg/mL solution. The mixture of cell lysis solution can be stored at −20°C for up to one year.

0.125% trypsin-EDTA solution

Reagent	Final concentration	Amount
0.25% trypsin-EDTA solution	50% (v/v)	25 mL
1× PBS	50% (v/v)	25mL
Total	N/A	50 mL

Note: Store at 4°C for up to three months.

Step-by-step method details

Design and construction of plasmids expressing pegRNAs and ngRNAs for prime editing

Timing: 5–8 days

This section outlines the design of pegRNA and ngRNA and their assembly into expression vectors for prime editing.

Note: The pegRNA and ngRNA components and their corresponding assembly workflows are summarized in Figure 2. Given that pegRNA performance is highly dependent on the sequence context of the target locus, and that no universally optimal computational platform can reliably identify the best-performing pegRNA across diverse loci and sequence contexts, this protocol employs multiple complementary design strategies to generate candidate pegRNAs for experimental evaluation.

• 1.Retrieve porcine APP, PSEN1, and TAU gene information from Ensembl database and define the desired edited sequence, as shown in Figure 1.
• 2.
  Design candidate pegRNAs using multiple strategies.

  • a.
    Use PrimeDesign³ (https://github.com/pinellolab/PrimeDesign) to design pegRNA1 for APP, PSEN1, and TAU. Select the top-ranked pegRNA proposed by the algorithm for each intended edit.
  • b.
    Design the second pegRNA (pegRNA2) for APP, PSEN1, and TAU using pegFinder² (http://pegfinder.sidichenlab.org/) and select the recommended pegRNA generated by the platform.
  • c.
    Design pegRNA3 for APP, PSEN1, and TAU using the PRIDICT 2.0 model⁴ (http://www.pridict.it/) and select the pegRNA with the highest predicted editing efficiency.
  • d.
    Manually tune pegRNA4 for each target according to recommended design rules derived from high-throughput pegRNA activity profiling studies.⁶

    • i.
      Initially set the primer binding site (pbs) and reverse transcription template (RTT) lengths to 13 nt and 12 nt.
    • ii.
      Adjust the pbs to 15 nt if its GC content is < 40%, or truncate it to 9-11 nt if the GC content is > 60%.
    • iii.
      Modulate the RTT so that its 3′ terminus preferably end with G or T and its 5′ end extends at least 5 nt beyond the intended edit site.

Note: PrimeDesign, pegFinder, and PRIDICT 2.0 are user-friendly platforms that provide recommended pegRNA sequences. Additional computational tools can also be evaluated when adapting this workflow to other loci, including the OPED⁷ and pegIT.⁸

• 3.Select ngRNAs that target the edited strand and are PE3b-compatible using PRIDICT 2.0 or PrimeDesign; if no PE3b-compatible candidates are identified, use the highest-scoring prediction by these tools.
• 4.Commercially synthesize the required oligonucleotide pairs with the designed overhangs shown in Figure 2, including sequences for the spacer, pegRNA extension, and sgRNA scaffold (see Tables S1 and S2).
• 5.For each oligonucleotide pair, prepare the annealing mix on ice using the following components. Heat the mixture in boiling water for 2 min, then allow it to cool gradually to 15°C–30°C to complete oligonucleotide annealing.

Reagent	Amount
Top oligonucleotide (100 μM)	2 μL
Bottom oligonucleotide (100 μM)	2 μL
10× T4 DNA ligase buffer	2.5 μL
ddH2O	18.5 μL
Total	25 μL

Figure 2.

Construction workflow for pegRNA and ngRNA expression plasmids

(A) Schematic overview of pegRNA plasmid construction. pegRNA components, including the spacer, sgRNA scaffold, and extension region, were designed using computational tools combined with manual optimization according to established pegRNA design principles. Corresponding oligonucleotides were synthesized, annealed and assembled into the BsaI-digested pU6-pegRNA-GG backbone via Golden Gate assembly to generate pegRNA expression plasmids.

(B) Schematic overview of ngRNA plasmid construction. ngRNA spacer sequences were designed in silico, followed by oligonucleotide synthesis and annealing. The annealed oligonucleotides were ligated into the BsaI-digested pGL3-U6-sgRNA-EGFP backbone using Golden Gate assembly to generate ngRNA expression plasmids.

Figure 1.

Schematic representation of the target loci and intended prime editing outcomes in APP, PSEN1, and TAU

Genomic regions flanking the prime editing target sites in porcine APP, PSEN1, and TAU are illustrated. For each locus, a zoomed-in view of the reference (WT) and intended edited sequences is displayed, with edited nucleotides highlighted in red. The corresponding amino acid substitutions resulting from prime editing—APP^K652N/M653L (GA>TC), PSEN1^M143L (A>C), and TAU^P300L (CA>TG)—are indicated below each sequence.

Note: After annealing, add 75 μL of distilled water to each reaction to dilute the annealed oligonucleotides. The diluted products are then used for subsequent pegRNA or ngRNA plasmid ligation.

• 6.For pegRNA assembly, prepare the reactions for each pegRNA plasmid on ice using the components listed below. Run the assembly reactions in a thermocycler with 7 cycles of 25°C for 15 min and 16°C for 5 min, followed by 37°C for 5 min and 80°C for 15 min, hold at 6°C until sample retrieval (Figure 2A).

Reagent	Amount
BsaI digested pU6-pegRNA-GG-acceptor vector	30 ng
Annealed spacer oligonucleotides	1 μL
Annealed extension oligonucleotides	1 μL
Annealed sgRNA scaffold oligonucleotides	1 μL
BsaI-HFv2	0.4 μL
T4 DNA ligase	0.5 μL
10× T4 DNA ligase buffer	1 μL
ddH2O	Up to 10 μL

• 7.For ngRNA assembly, prepare the reactions for each ngRNA plasmid on ice using the components listed below. Incubate at 15°C–30°C for 15 min to ligate the annealed ngRNA oligonucleotides into the linearized ngRNA backbone (Figure 2B).

Reagent	Amount
BsaI digested pGL3-U6-sgRNA-EGFP vector	30 ng
Annealed spacer oligonucleotides	2 μL
T4 DNA ligase	0.5 μL
10× T4 DNA ligase buffer	1 μL
ddH2O	Up to 10 μL

• 8.
  Transform and verify ligated plasmids, following the workflow shown in Figure 3.

  • a.
    Transform the ligated plasmids into E. coli DH5α competent cells.
  • b.
    Pick single colonies after 14 h of bacterial culture.
  • c.
    Confirm correct insert sequences by Sanger sequencing.

Note: See problem 1 for troubleshooting.

• 9.Purify plasmid DNA using the TaKaRa MidiBEST Endo-free Plasmid Purification Kit.

CRITICAL: Endo-free plasmid is essential for efficient PFF transfection.

Pause point: Sequence-verified, endotoxin-free pegRNA and ngRNA plasmids can be stored at −20°C for several months before use in electroporation experiments.

Figure 3.

Workflow diagram for plasmid transformation, clone screening, and plasmid preparation

A schematic overview of the workflow used for pegRNA/ngRNA plasmid transformation and validation. After transformation and plating, individual colonies are picked following a 14 h incubation and verified by Sanger sequencing. Sequence-confirmed clones are subsequently amplified and subjected to plasmid extraction.

Evaluation of candidate pegRNA/ngRNA pair activities in PFFs

Timing: 15–25 days

The following procedure outlines screening of candidate pegRNA/ngRNA pairs in PFFs to identify combinations with the highest editing activity for each target locus, following the workflow outlined in Figure 4.

• 10.
  Preparation of PFFs for electroporation.

  • a.
    Preheat the regular growth medium in a 37°C water bath for 15 min.
  • b.
    Retrieve a cryovial of PFFs from liquid nitrogen and thaw the cells in a 37°C water bath with gentle swirling until the ice crystals disappear.
  • c.
    Immediately transfer the contents to a sterile 15 mL conical tube containing 5 mL of pre-warmed regular growth medium.
  • d.
    Centrifuge at 200 × g for 5 min at 15°C–30°C, gently remove the supernatant, and resuspend the cell pellet in 10 mL of regular growth medium.
  • e.
    Seed the cells into a 10-cm culture dish and incubate at 37°C in a humidified incubator with 5% CO₂.
  • f.
    Monitor cell morphology and confluence daily under a microscope until the cells reach 85%–90% confluence.
  • g.
    Wash cells once with 8 mL 1× PBS and gently aspirate the buffer.
  • h.
    Add 1 mL of 0.125% trypsin-EDTA solution and incubate at 37°C for 3–4 min.
  • i.
    Add 7 mL of fresh growth medium to the dish to inactivate trypsin.
  • j.
    Gently pipette to detach any remaining adherent cells, then transfer the cell suspension to a sterile 15 mL conical tube.
  • k.
    Centrifuge at 200 × g for 5 min, discard the supernatant, and resuspend in pre-warmed regular growth medium.
  • l.
    Seed cells evenly into three new 10-cm dishes, continue culturing the cells until they reach 85%–90% confluence for subsequent electroporation.

Note: At 85%–90% confluence, each 10-cm dish contains approximately 3–3.5 × 10⁶ cells.

• 11.
  Electroporation and enrichment of edited PFFs.

  • a.
    Prepare the following plasmid master mix for a single electroporation reaction.

    Plasmid	Amount
    pvPE-V3-Puro	1 μg
    pegRNA	0.5 μg
    ngRNA	0.5 μg

    CRITICAL: Measure each plasmid stock concentration before mixing to ensure accurate and reproducible downstream experiments.

  • b.
    Confirm that the PFFs prepared in step 10 are 85%–90% confluent before electroporation.
  • c.
    Warm regular growth medium and 1× PBS in a 37°C water bath for 15 min. Meanwhile, pre-incubate the Entranster-E reagent in a 37°C CO₂ incubator.
  • d.
    Wash PFFs once with 8 mL of pre-warmed 1× PBS per dish and discard the PBS. Detach the cells with 1 mL of 0.125% trypsin-EDTA solution at 37°C for 3 min.
  • e.
    Resuspend detached cells in fresh growth medium and transfer to 15 mL sterile centrifuge tubes.
  • f.
    Count the cells and pool the required number (4 × 10⁵ PFFs per reaction) into a 15 mL tube. Centrifuge at 200 × g for 5 min at 15°C–30°C.
  • g.
    Carefully remove the supernatant and resuspend the cell pellet in 20 μL Entranster-E reagent per electroporation reaction.

    Note: Thorough removal of residual medium is critical, as liquid carryover can dilute the Entranster-E/cell mixture and reduce electroporation efficiency. After centrifugation, carefully aspirate the supernatant by placing a sterile pipette tip against the tube wall. To remove residual droplets without disturbing the cell pellet, gently tilt and rotate the tube while repeating aspiration 2–3 times. For multiple electroporation reactions, pool the required number of cells prior to centrifugation to minimize residual liquid carryover.

  • h.
    Add 20 μL cell suspension to each tube containing the plasmid master mix and gently pipette up and down to mix uniformly.
  • i.
    Transfer the cell-DNA mixture into each well of 4D-Nucleofector™ strip and electroporate using the customized CA-137 + CA-137 dual-program.⁹
  • j.
    Following electroporation, transfer each electroporation mixture into a well of a 6-well plate containing 2 mL pre-warmed growth medium and incubate under standard conditions (37°C, 5% CO₂).
  • k.
    Aspirate the medium at 8 h post-electroporation and replace with growth medium containing 500 ng/mL nocodazole; continue culturing the cells.

    Note: See problem 2 for troubleshooting.

  • l.
    After 40 h of nocodazole treatment, replace the medium with fresh growth medium containing 2 μg/mL puromycin.
  • m.
    Incubate for 24 h to enrich transfected cells, then switch to puromycin-free medium for recovery.

    Note: Nocodazole and puromycin are applied sequentially rather than simultaneously. Nocodazole is introduced during the active editing window to modulate the cell cycle and enhance prime editing efficiency. Puromycin is applied afterward, once sufficient expression of the puromycin-resistance cassette has been achieved to enrich transfected cells. This sequential strategy also helps reduce potential combined cytotoxicity.

• 12.
  Editing efficiency analysis.

  • a.
    Genomic DNA preparation.

    • i.
      After 24 h of recovery, add 150 μL of 0.125% trypsin-EDTA solution per well. Gently rock the plate to ensure even coverage of the cells and incubate at 37°C for 3 min.
    • ii.
      Examine the cells under a microscope to confirm detachment; if detachment is incomplete, extend the incubation for an additional 1–2 min as needed.
    • iii.
      Add 100 μL of fresh growth medium to terminate digestion, then transfer 200 μL or the entire cell suspension into 0.2 mL tubes.

      Note: Use 0.2 mL 8-tube strips to streamline downstream lysis and PCR setup. Typically, 0.2 mL 8-tube strips can accommodate a working volume of up to approximately 250 μL.

    • iv.
      Centrifuge the cells at 200 × g for 5 min; carefully remove the supernatant, and add 12–20 μL of cell lysis solution per tube.

      Note: Avoid disturbing the cell pellet when aspirating the supernatant.

    • v.
      Lyse the cells using prepared cell lysis solution in a thermocycler at 56°C for 2.5 h, followed by 85°C for 20 min.
  • b.
    First-round PCR amplification and Sanger sequencing.

    • i.
      Prepare the following reactions on ice.

      Reagent	Amount
      Genomic DNA from edited PFFs	1 μL
      PrimeSTAR® Max DNA Polymerase (2×)	14 μL
      Forward primer (10 μM)	1.2 μL
      Reverse primer (10 μM)	1.2 μL
      ddH2O	10.6 μL
      Total	28 μL

      Note: Design high-quality primers (see Table S3) that amplify the intended editing site with >200 bp flanking sequence on each side to meet the coverage requirements for second-round PCR/NGS analysis.

    • ii.
      Then initiate PCR reactions using the following program.

      Steps	Temperature	Time	Cycles
      Pre-denaturation	95°C	5 min	1
      Denaturation	95°C	30 s	26 cycles
      Annealing	68–55°C, −0.5°C/cycle	30 s
      Extension	72°C	1 min
      Denaturation	95°C	30 s	14 cycles
      Annealing	55°C	30 s
      Extension	72°C	1 min
      Final extension	72°C	5 min	1
      Hold	6°C	∞

    • iii.
      Perform Sanger sequencing of the PCR products using a commercial sequencing service.
    • iv.
      Analyze Sanger sequencing chromatograms using SnapGene or equivalent software to assess editing outcomes, as shown in Figure 5A.

      Optional: If the pegRNA/ngRNA pair yields clearly detectable on-target editing peaks in the Sanger trace, proceed directly to monoclonal line derivation to shorten the workflow.

      Note: When Sanger traces show weak or indistinct editing peaks, or when relative peak heights prevent clear ranking of pegRNA/ngRNA pairs, use next-generation sequencing (NGS) to quantify editing efficiency and identify the best combination.

  • c.
    Second-round PCR amplification and next-generation sequencing (NGS):

    • i.
      Prepare the following reactions on ice.

      Reagent	Amount
      First-round PCR product	0.4 μL
      PrimeSTAR® Max DNA Polymerase (2×)	14 μL
      Forward primer (10 μM)	1.2 μL
      Reverse primer (10 μM)	1.2 μL
      ddH2O	11.2 μL
      Total	28 μL

    • ii.
      Initiate second-round PCR reactions using the cycling program (refer to step 12b).
    • iii.
      Submit the second-round PCR products to a commercial NGS provider for targeted amplicon sequencing.

      Note: Paired-end sequencing is recommended to ensure sufficient coverage of the intended editing site. For each sample, at least 5,000 demultiplexed reads are recommended for editing-efficiency assessment.

  • d.
    NGS data analysis.

    • i.
      Download demultiplexed FASTQ files from the commercial sequencing provider and analyze the reads using CRISPResso2⁵ in ‘Prime-Editing’ mode.

      Note: Specify the following required inputs: reference amplicon sequence, pegRNA spacer sequence, pegRNA extension sequence, pegRNA scaffold sequence, and nicking guide sequence. Keep the remaining parameters at their default settings unless locus-specific adjustment is needed.

    • ii.
      Based on the CRISPResso2 output, evaluate the intended editing efficiency and byproduct frequencies.

      Note: Reads classified as ‘Prime-edited UNMODIFIED’ are counted as intended edits, whereas all other reads are counted as byproducts, including ‘reference MODIFIED’, ‘Prime-edited MODIFIED’, ‘AMBIGUOUS’, and ‘Scaffold-incorporated’.

    • iii.
      Visualize the quantified results using GraphPad Prism 9.5 or equivalent software, as illustrated in Figure 5B.
    • iv.
      Compare the editing outcomes across all pegRNA/ngRNA combinations and select the optimal pair based on high intended editing efficiency and low levels of byproduct.

      Note: See problem 3 for troubleshooting.

Figure 4.

Screening of high-activity pegRNA/ngRNA pairs in PFFs

Workflow for evaluating pegRNA/ngRNA activity in PFFs. Cells are electroporated with pvPE and pegRNA/ngRNA constructs, followed by nocodazole treatment and puromycin selection. Genomic DNA is prepared by cell lysis and subjected to first- and second-round PCR. Sequencing-based analysis is used to identify high-activity pegRNA/ngRNA pairs for downstream applications. FP: forward primer; RP: reverse primer.

Figure 5.

Identification of the optimal pegRNA/ngRNA pair for each target in PFFs

(A) Representative Sanger sequencing traces showing prime editing outcomes mediated by individual pegRNA/ngRNA pairs (Pairs 1–4) targeting APP, PSEN1, and TAU in PFFs. The targeted nucleotides are highlighted by black boxes.

(B) Quantification of prime editing efficiencies and indel formation for each pegRNA/ngRNA pair targeting APP, PSEN1, and TAU in PFFs.

Editing efficiency was calculated as the percentage of reads carrying the intended edit among total aligned reads. Data are presented as means ± standard deviations with three independent biological replicates. WT (wild-type) cells, transfected without pegRNA/ngRNA, served as negative control.

Generation of multiplexed prime-edited PFF monoclonal cell lines

Timing: 27–35 days

The protocol below details the workflow for establishing PFF monoclonal cell lines carrying the multiplexed prime-edited AD-related pathological mutations (APP^K652N/M653L, PSEN1^M143L, and TAU^P300L).

Note: In this workflow, the pvPE system is employed with the best-performing pegRNA/ngRNA pairs identified in the preliminary screening. An overview of the workflow is shown in Figure 6.

• 13.
  Electroporation of PFFs.

  • a.
    Prepare the following plasmids master mix on ice.

    Plasmid	Amount
    pvPE-V3-Puro	5 μg
    APPK652N/M653L-pegRNA	2.5 μg
    APPK652N/M653L-ngRNA	2.5 μg
    PSEN1M143L-pegRNA	2.5 μg
    PSEN1M143L-ngRNA	2.5 μg
    TAUP300L-pegRNA	2.5 μg
    TAUP300L-ngRNA	2.5 μg

    CRITICAL: Use high-concentration plasmids (e.g., 2–3 μg/μL) so that the total plasmid volume remains below 10% of the electroporation buffer volume.

  • b.
    Harvest early-passage PFFs at 85%–90% confluence with 0.125% trypsin-EDTA, then resuspend the detached cells in regular growth medium (refer to steps 10h-i for details).

    Note: Use early-passage PFFs to maintain high viability and improve colony formation efficiency.

  • c.
    Count the cells and aliquot 2 × 10⁶ cells into a 15 mL tube, then centrifuge at 200 × g for 5 min at 15°C–30°C.
  • d.
    Carefully and thoroughly aspirate the supernatant, and resuspend the cell pellet in 100 μL of Entranster-E transfection reagent per reaction.
  • e.
    Transfer the mixture into the Lonza 4D-Nucleofector™ cuvettes and electroporate using the customized CA-137 + CA-137 dual-program.⁹
  • f.
    Gently transfer the electroporated cells into a 10-cm dish containing 10 mL pre-warmed growth medium, adding dropwise while swirling the dish to ensure even distribution.
  • g.
    After 8 h, replace the medium with fresh growth medium containing 500 ng/mL nocodazole.

    Note: Morphological changes after nocodazole treatment are largely reversible upon drug removal.

  • h.
    After 48 h, replace medium with fresh growth medium supplemented with 2.5 μg/mL puromycin. Continue culturing for an additional 48 h.

    Note: See problem 4 for troubleshooting.

• 14.
  Limiting-dilution cloning and colony formation.

  • a.
    Harvest PFFs by incubating with 0.125% trypsin-EDTA for 3–4 min at 37°C.
  • b.
    Neutralize the reaction by adding 10 mL clonal growth medium, and gently pipette the cell suspension to ensure even dispersion.
  • c.
    Transfer 50 μL, 100 μL, 150 μL, 200 μL and 250 μL aliquots of the cell suspension into separate 10-cm dishes containing 10 mL of clonal growth medium to identify the optimal seeding density.

    CRITICAL: Under 100× microscope (10× objective, 10× eyepiece), a density of ∼2 cells per field serves as a reference.

  • d.
    Based on this reference, seed the remaining dishes across a gradient of cell densities, ranging from slightly below to slightly above the reference density, to maximize the likelihood of obtaining well-spaced single-cell colonies.

    Note: To ensure an adequate yield of monoclonal colonies, plate 10–15 10-cm dishes for each density level.

  • e.
    Refresh clonal growth medium every 4–5 days, and monitor individual colonies for size and spatial separation to assess clonal growth.
• 15.
  Isolation of monoclonal colonies, as illustrated in Figure 7.

  • a.
    When individual colonies become macroscopically distinguishable through the bottom of the dish, carefully outline each clone with an indelible marker.
  • b.
    Pre-warm clonal growth medium and 1× PBS.
  • c.
    Remove the lids from sterile 1.5 mL tubes using sterile scissors. At the same time, dispense a small amount of sterile vaseline into a 10-cm dish for later sealing.
  • d.
    Pre-fill the 24-well plates with 1.5 mL clonal growth medium per well.
  • e.
    Gently rinse each 10-cm dish with 8 mL 1× PBS and aspirate completely.
  • f.
    Dip the cut rim of a 1.5 mL tube into vaseline, and draw a thin, continuous ring of vaseline around each marked colony.

    CRITICAL: Each colony must be completely enclosed by an unbroken vaseline ring; any gap will let reagents leak beyond the marked area and compromise colony isolation.

  • g.
    Carefully add 50 μL of 0.125% trypsin-EDTA solution to each enclosed colony. After 40 s of digestion, remove the solution and incubate the dishes for an additional 4 min at 37°C.
  • h.
    Neutralize trypsin by adding 50 μL of fresh clonal growth medium to each colony, gently pipette to detach the cells.
  • i.
    Transfer each individual clone into a separate well of a 24-well plate pre-filled with clonal growth medium.
  • j.
    Culture the clones in 24-well plates until they reach approximately 85%–90% confluence.
  • k.
    Detach the clones by trypsinization and transfer the cell suspension into an individual well of 6-well plates containing 2 mL of clonal growth medium.
• 16.
  Genotyping of monoclonal colonies:

  • a.
    When clones reach approximately 85%–90% confluence, harvest 10% of each clone by trypsinization.

    Note: The harvested cells from the previous step will serve as templates for the first-round PCR amplification of the target site. Insufficient confluence may result in inadequate cell numbers for subsequent experimental steps, whereas excessive confluence can lead to aberrant morphology and compromise clone quality.

    Note: See problem 5 for troubleshooting.

  • b.
    Transfer the remaining cells from each clone into a 6-cm dish containing 5 mL clonal growth medium.
  • c.
    Amplify the target loci using the first-round PCR conditions described in step 12b. Submit the amplicons for Sanger sequencing to genotype the edited clones, as shown in Figure 8A.
  • d.
    Identify successfully edited single-cell colonies of the target genes by Sanger sequencing results, as shown in Figure 8B.
  • e.
    Continue culturing the desired monoclonal cell lines until they reach 85%–90% confluence.

Figure 6.

Experimental workflow for the generation of monoclonal gene-edited PFF cell lines

Schematic of the experimental pipeline. After electroporation with prime-editing vectors, cells recover for 8 h and are then treated with nocodazole to enhance editing efficiency. Puromycin selection follows to enrich transfected cells. Subsequently, cells are subjected to single-cell dilution to form colonies. Individual monoclonal colonies are isolated and validated by PCR and sequencing to confirm genomic modifications.

Figure 7.

Identification and isolation of single-cell-derived colonies

Representative images of single-cell colony recognition. Colonies are visually examined, marked on the bottom of the dish, and circled with a 1.5 mL tube (tip removed) coated with vaseline to guide manual picking.

Figure 8.

Genotypic screening and validation of triple-locus edited monoclonal clones

(A) Heatmap summary of the genotypic distribution across 60 single-cell-derived clones at three targeted loci (APP, PSEN1, and TAU). Genotypes are categorized as wild type (WT, 0), heterozygous (He, 1), or homozygous (Ho, 2), indicated by the color gradient from light pink to dark red. Clones #19 and #38 are highlighted as representative lines carrying successful edits at multiple loci.

(B) Representative Sanger sequencing chromatograms confirming the precision of genomic modifications. Comparison of wild type (WT) sequences against edited clones (#19 and #38) demonstrates the targeted base substitutions: GA to TC (APP^K652N/M653L), A to C (PSEN1^M143L), and CA to TG (TAU^P300L). Black boxes highlight the specific locations of the induced mutations.

Expected outcomes

Using the pvPE system, this protocol yields PFF monoclonal cell lines carrying multiplexed prime editing-mediated modifications. During initial screening, target loci are PCR-amplified and analyzed by Sanger or next-generation sequencing to assess individual pegRNA/ngRNA combinations, thereby identifying high-efficiency pairs for each target gene (Figure 5).

After single-cell cloning, correctly edited PFF monoclonal cell lines harboring precise APP^K652N/M653L, PSEN1^M143L, and TAU^P300L mutations in three Alzheimer’s disease-associated genes can be validated by genotyping (Figure 8). Collectively, these results demonstrate that the protocol enables efficient multiplexed prime editing and the establishment of PFF monoclonal cell lines.

Limitations

This protocol requires experienced personnel in a well-equipped mammalian cell culture facility. Poor aseptic technique or failure to control cell density during clonal isolation can lead to contamination or inefficient single-cell outgrowth, reducing overall reproducibility.

In addition, pegRNA/ngRNA activity is highly context-dependent. Although this protocol includes systematic screening to identify high-activity pegRNA/ngRNA pairs, the design of efficient pegRNAs remains challenging and might require iterative optimization for specific genomic loci. In particular, the availability of suitable PAM sequences and constraints on spacer design can limit editing efficiency at certain target sites. For example, at the PSEN1 locus examined in this study, the limited availability of suitable SpCas9-compatible PAM sequences near the desired edit constrained pegRNA design and likely contributed to the reduced editing efficiency.

Troubleshooting

Problem 1

Few or no correctly assembled pegRNA plasmids are obtained after colony screening. (Step 8).

Potential solution

Low pegRNA plasmid assembly efficiency can result from incompatible oligonucleotide design, incomplete backbone digestion, or inefficient ligation of annealed components. Verify that all spacer, sgRNA scaffold, and pegRNA extension sequences contain the correct overhangs and lack internal BsaI recognition sites. If correct clones are rare and many wild-type or empty-backbone colonies are recovered, ensure complete digestion and purification of the pU6-pegRNA-GG backbone before assembly. Increasing BsaI-HFv2 to 0.8 μL in the pegRNA plasmid assembly master mix may also improve assembly efficiency. Additionally, ensure that both sgRNA scaffold oligonucleotides are 5′ phosphorylated to enable efficient ligation.

Problem 2

A substantial amount of cell death was observed during the subsequent medium exchange after electroporation. (Step 11k).

Potential solution

Increase the cell number per reaction and reduce the total amount of plasmid DNA to improve post-electroporation viability. Additionally, test alternative electroporation programs with higher survival rates (e.g., EN-150 or CA-137 single-programs).

Problem 3

All tested pegRNA/ngRNA pairs exhibit consistently low editing efficiency (Step 12d).

Potential solution

Consider first testing enhanced prime editor variants, such as pvPE-V4, to improve baseline editing efficiency. In addition, because mismatch repair (MMR) can reduce prime editing efficiency in some contexts, MMR-modulating strategies, such as overexpression or fusion of dominant-negative MLH1 (MLH1dn)¹⁰ or an MLH1-targeting small binder,¹¹ may be considered as advanced optimization options. If editing efficiencies remain low, redesign and clone additional pegRNAs using alternative spacers, as spacer choice is a major determinant of pegRNA activity.^6,12 When possible, include at least two spacers per target in the initial screening panel. Alternatively, use prime editor systems based on nCas9 variants with altered PAM specificities to expand targetable loci, such as SpCas9-NG (NG PAM),¹³ SpG (NG/NGN PAM),¹³ SpRY (near-PAMless targeting),¹³ or SaCas9-based PE systems (NNGRRT or engineered NNNRRT PAMs).¹⁴ These alternatives improve targeting accessibility when canonical SpCas9-compatible PAMs are limited.

Problem 4

Cell viability is markedly reduced after treatment with nocodazole and puromycin (Steps 11l and 13h).

Potential solution

Reduce the concentrations and/or duration of nocodazole and puromycin treatment, and extend the recovery period before downstream processing. In our optimized PFF workflow, treatment with 500 ng/mL nocodazole for 40 h was feasible. However, if obvious cytotoxicity or poor post-treatment recovery is observed, milder conditions may be used, such as reducing nocodazole to 100 ng/mL, shortening the treatment duration to 24–32 h, or extending the recovery period after drug removal. For puromycin selection, the concentration and/or duration should be reduced, if excessive cell death occurs. For example, puromycin may be decreased from 2 μg/mL to 1–1.5 μg/mL, or the selection period may be shortened. Additionally, supplementation of the growth medium with basic fibroblast growth factor (bFGF, 2.5 ng/mL) may further enhance cell proliferation and facilitate recovery after treatment-induced stress.

Problem 5

Incomplete trypsin digestion results in poor cell detachment, whereas prolonged digestion compromises cell viability (Step 16a).

Potential solution

This troubleshooting strategy can also be applied to Steps 15g, 15k, and 16a, in which single-cell-derived clones are not sufficiently detached due to suboptimal digestion. Before large-scale digestion, perform a preliminary experiment to establish the optimal digestion time for your handling conditions. Start with 0.125% trypsin and monitor cell morphology under a microscope to determine the appropriate digestion time. If efficiency remains low, increase the trypsin concentration to 0.25% and further optimize the digestion duration as needed. In addition, thoroughly wash cells with PBS before digestion; residual serum-containing medium can inhibit trypsin activity and reduce efficiency.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Weiwei Liu (wwliu199@outlook.com).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be fulfilled by the technical contact, Yuyun Xing (xingyuyun9@hotmail.com).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and code availability

This study did not generate/analyze new datasets or code.

Acknowledgments

We would like to thank Prof. Lusheng Huang from Jiangxi Agricultural University for his guidance in this research. This work was supported by the grant from the National Natural Science Foundation of China (grant no. 32260825) and the National Key Research and Development Program of China (grant no. 2023YFC3404302).

Author contributions

W.L. and Y.X. designed the project. W.D., W.L., and Y.X. wrote the article. W.L. designed the sequences of pegRNA and ngRNA plasmids. W.D. and W.L. performed the electroporation of PFF cells and the generation of desired PFF cell lines.

Declaration of interests

The authors have filed a patent application related to the findings of this study.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT, developed by OpenAI, to assist in improving the readability and language clarity of the manuscript. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.