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Method Article

Generation of Genetically Engineered Embryos Using Gene Editing and Somatic Cell Nuclear Transfer for Production of Sheep Models of Human Disease

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DOI:

10.3791/72709

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September 15th, 2026

 ,  ,  ,  ,  , 
1Department of Animal, Dairy, and Veterinary Sciences, Utah State University

In This Article

Summary

Here, we describe an approach to produce genetically modified embryos using CRISPR-Cas9 gene editing and somatic cell nuclear transfer for generating sheep models of human disease. This approach is ideal for precise gene editing in donor cells and producing cloned embryos without genetic mosaicism.

Abstract

Large animal models are valuable tools for investigating human disease. Sheep, pigs, and goats often better recapitulate the anatomy and physiology of human organs and the complexity of human disease, thereby enhancing their clinical relevance compared to rodents. CRISPR-Cas9 and somatic cell nuclear transfer (SCNT) enable the generation of large animal models with greater precision, versatility, and genetic uniformity. The primary benefit of this approach, compared with zygote microinjection, is the ability to confirm in vitro whether the desired genetic modification and potential off-target mutations are present in gene-edited cells prior to animal production. Moreover, SCNT eliminates the chance of genetic mosaicism, which frequently results from zygote microinjection. Here, we describe the generation of gene-edited ovine cells through non-homologous end-joining (NHEJ) and homology-directed repair (HDR), followed by the production of cloned embryos carrying the mutations of interest. Genetic modifications are introduced by transfecting cultured somatic cells, typically fetal fibroblasts, with the CRISPR-Cas9 system. Mutation efficiency in pooled cells is assessed by polymerase chain reaction (PCR) and Sanger sequencing of edited genes and analyzed using Tracking of Indels by DEcomposition (TIDE)/Tracking of Insertions, Deletions, and Recombination events (TIDER) software. Limiting dilution of the pooled cells is performed to obtain single-cell-derived colonies, which are screened by PCR and DNA sequencing of edited genes. Donor cells with the edit(s) of interest are subsequently expanded and used for the generation of embryos by SCNT. After limiting dilution and cell screening, 22/114 (19.3%) of colonies modified through NHEJ contained knockout (KO) mutations and 4/56 (7.1%) of colonies modified with HDR contained the F508del mutation. A total of 370 genetically modified embryos were created from four colonies. These methods are successfully used for precise gene editing in fetal fibroblasts and generation of genetically engineered embryos to produce ovine models of human disease.

Introduction

Animal models are essential tools for understanding the biological mechanisms of human diseases and development of new therapeutics. In vitro models, such as cell or organoid culture, do not reproduce the complexity of cellular subtypes and cell-cell interactions that occur in vivo in organs1,2. Thus, animals provide whole-organism models with greater detail of cellular and system-level interactions, disease progression, and the safety of therapeutics. Rodents are practical model organisms given their low cost, quick generation time, and comprehensive genetic characterization. However, rodent models for human diseases are limited by physiological and anatomical differences with humans, as well as their faster metabolism and shorter lifespans3,4. This is evidenced by the high proportion of therapies tested in animal models that fail in clinical trials, the majority of which use rodents5,6,7,8. Sheep, pigs, goats, dogs, and non-human primates have been used to produce models for a wide variety of human diseases, which may help increase the clinical translation rate for new therapies and medicines3,9,10,11,12. These species also offer many advantages over rodent models due to their increased similarity to humans. Large animal models for human disease thus provide the most accurate models available and play a critical role in translating animal studies into clinical applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a versatile and programmable method for genetic modification of animal cells and introduction of precise disease-causing mutations. CRISPR-Cas9-mediated gene editing employs a guide RNA (gRNA) complementary to the target site, allowing CRISPR-associated protein 9 (Cas9) to form a double-stranded break (DSB) in a targeted manner13. Cellular repair of DSBs occurs primarily through two mechanisms: non-homologous end joining (NHEJ) and homology-directed repair (HDR). The NHEJ pathway repairs a DSB by joining DNA ends, an error-prone mechanism that may result in the formation of random insertions or deletions (indels) at the break site14. These mutations may knock out (KO) the gene by creating a frameshift mutation or a premature stop codon, which disrupt the gene’s function. HDR, occurring mainly during the S and G2 phases of the cell cycle, is when the cell repairs a DSB using a homologous allele as a repair template15,16. Incorporation of specific insertions, deletions, or substitutions, known as knock-ins (KI), is possible by introducing a synthetic single-stranded oligodeoxynucleotide (ssODN) or other template DNA with the edit of interest, which the cell can use to repair the break. CRISPR-Cas9 may also be used for multiplexed editing to introduce multiple mutations at once for modeling multigenic diseases3. Compared to previous gene-editing techniques such as zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR-Cas9 is much simpler and more resource efficient to use in large animal cells17. Different loci can be targeted easily by designing a new gRNA, which has helped accelerate the introduction of new large animal models using CRISPR-Cas93,13. However, several limitations remain, including the editing efficiency of CRISPR-Cas9, which can vary considerably depending on the species, cellular environment, and nature of the edit18,19. HDR rates, in particular, are lower than NHEJ rates and depend greatly on the cell type, genomic loci, and cell cycle stage20,21. Moreover, the introduction of unintended edits at off-target loci, or even on-target loci, remains an issue22. Even so, CRISPR-Cas9 has become the primary gene editing platform for producing genetically engineered large animal models.

Somatic cell nuclear transfer (SCNT) facilitates the creation of genetically modified embryos with high precision and homogeneity. This process, first successfully performed in mammals in 1997, involves the transfer of a cell nucleus into an enucleated oocyte23. SCNT possesses many advantages over other techniques, such as zygotic microinjection, in which genetic material or DNA-editing systems are directly injected into the zygote or early embryo24,25,26. Zygotic microinjection carries the inherent risk of genetic heterogeneity, or mosaicism, between cells in the embryo, which can occur if DNA replication or cell division occurs prior to genetic modification27,28,29. The value of such mosaic embryos for animal models is reduced due to the difficulty of genotyping and phenotypic characterization29. In SCNT, gene editing is decoupled from embryo production; each cloned embryo is derived from a single cell, eliminating the risk of mosaicism and ensuring the embryo is genetically homogeneous30,31,32. Additionally, donor cells are edited and carefully screened in vitro to confirm clonality and the presence of the desired modification prior to the SCNT procedure, saving time and resources25,26. This is especially important when introducing specific KIs. Cell screening also allows the identification of potential mutations in off-target sites, thus reducing the risk of genetic disruption at other loci. These advantages address many of the concerns associated with CRISPR-Cas9. Therefore, SCNT provides greater control over the genetics of the resulting embryos compared to zygote microinjection, especially for more complex edits such as multiplexed edits, large insertions, and humanized alleles33. Although, SCNT is limited by low cloning efficiency, technical requirements, and abnormal epigenetic reprogramming, the latter of which can lead to molecular and phenotypic disruptions in clones, potentially obscuring the effects of genetic modification in founder animals. However, these epigenetic alterations are not passed on to the offspring32,33. Despite these limitations, the combination of SCNT with CRISPR-Cas9 enables precise genome engineering with strict control over embryo genetics, supporting the reliable production of large animal disease models without mosaicism. Here, we describe a detailed approach for genetic modification of sheep fetal fibroblasts (SFF) using either NHEJ or HDR, followed by SCNT for cloned embryo production, with the ultimate goal of generating sheep models of human disease. This approach is largely applicable to other livestock species, though gene editing may require optimization in different cell types, with this protocol acting as a useful starting point.

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Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC: #15508 and #10089) at Utah State University and performed in accordance with National Institutes of Health (NIH) guidelines. In this study, domestic Romney sheep (Ovis aries) were used to isolate sheep fetal fibroblasts (Supplementary File 134). See Figure 1 for an overview of the protocol.

Gene editing process diagram: PCR primer design, CRISPR transfection, genome editing analysis, cloning.
Figure 1: Overview of CRISPR-Cas9 and SCNT methods used to generate genetically modified embryos. A summary of each of the seven major steps of the protocol is depicted. Created in BioRender. Perisse, I. (2026). Please click here to view a larger version of this figure.

1. Design of PCR primers, gRNA, and template ssODN

  1. Open the genomic region to be edited in Benchling (https://www.benchling.com/academic).
  2. Design PCR primers to amplify and sequence the genomic region of interest.
    1. Highlight the target region to be amplified, plus 200 base pairs upstream and downstream of the target region to ensure ample distance for quality trace calls and accurate analysis of mutation efficiency with TIDE software.
    2. On the right-hand navigation bar click Primers > Create Primers > Wizard.
    3. Under Region, click Use Selection. Under Amplicon, adjust Size accordingly and adjust any other parameters as desired.
    4. Click Generate Primers. Select one or more primer pairs with low penalty scores to order.
      ​NOTE: Depending on the edit being introduced, it is recommended to design primers that will bind only to correctly edited DNA to allow for quicker screening of positive colonies.
  3. Design gRNA to edit DNA with CRISPR-Cas9.
    1. In Benchling, highlight the target region to be edited, then on the right-hand navigation bar, click CRISPR > Design and analyze guides.
    2. Click the Genome dropdown menu and select or import the model organism genome. Adjust any other parameters as desired.
    3. Click Finish > +.
    4. Under Genome Region, click No region set and select or input the genome region containing the target site. Click Set genome region.
    5. Select one or more guides with both high on-target scores and high off-target scores to order.
  4. If performing KI edits, design an ssODN template.
    1. On the right-hand navigation bar, click CRISPR > Design HR template (ssODN).
    2. Click the Genome dropdown menu and select or import the model organism genome. Click Create.
    3. Click on the SEQUENCE MAP tab, highlight the bases to be edited, and create the modified version of the DNA sequence by typing the desired edits into the keyboard. Go back to the DESIGN HR TEMPLATE tab and click Next.
    4. Adjust the Template region to include at least 50 base pairs of homology arms on each side of the edit, then click Next.
    5. Input the sequence of the gRNA selected in step 1.3.5 in the Guide box to introduce Protospacer Adjacent Motif (PAM)-disrupting silent mutations. Click Next.
    6. Click Copy the template and order the ssODN template generated.
      NOTE: Sequences of PCR primers, CRISPR-Cas9 gRNAs, and template ssODNs designed using Benchling for the generation of a sheep cystic fibrosis model carrying either a knockout (KO) mutation or the F508del mutation are presented in Table 1.

2. Media preparation

  1. Prepare cell culture medium by supplementing Dulbecco’s modified Eagle’s medium (DMEM) high Glucose with 15% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (Pen/Strep) (v/v). Pre-warm in a water bath at 37 °C before use.
  2. Prepare freezing medium by supplementing DMEM with 45% FBS and 10% dimethyl sulfoxide (DMSO).
  3. Prepare oocyte collection medium by supplementing M199 medium with 1% Pen/Strep, 0.3% bovine serum albumin (BSA) fraction V, 1% FBS, and 0.06% heparin sodium salt.
  4. Order In Vitro Maturation (IVM) and In Vitro Culture (IVC) media, which are commercially available serum-free media formulated for bovine oocyte maturation and embryo culture, respectively.
    NOTE: Post-fusion and IVC dishes are prepared with seven 40 µL drops of IVC medium and covered with embryo-tested mineral oil.
  5. Prepare cell starvation medium by supplementing DMEM high Glucose with 0.5% FBS and 1% Pen/Strep (v/v). Pre-warm in a water bath at 37 °C before use.
  6. Prepare HEPES-supplemented synthetic oviductal fluid (HSOF) medium as previously described35.
  7. Prepare enucleation medium by supplementing 500 µL of HSOF medium with 4.5 mM sucrose and 10 µg/mL Cytochalasin B (CB).
  8. Prepare injection media by supplementing 500 µL of HSOF medium with 10 µg/mL Cytochalasin B (CB).
    NOTE: Enucleation and injection media are prepared fresh on the day of SCNT. One drop (200 µL) of each medium is placed in 100 mm dishes and covered with embryo-tested mineral oil.
  9. Prepare fusion medium as previously described35. Remove 5–10 mL of fusion medium from 4 °C and keep at room temperature until use.
  10. Prepare CB Medium by supplementing IVC medium with 10 µg/mL CB.
    NOTE: A CB dish is prepared with seven 40 µL drops of CB medium and covered with embryo-tested mineral oil.
  11. Prepare the ionomycin medium by supplementing the HSOF medium with 5 µM ionomycin calcium salt.
  12. Prepare activation medium by supplementing IVC medium with 2 mM 6-Dimethylaminopurine (DMAP) and 10 µg/mL Cycloheximide (CHX).
    NOTE: An activation dish is prepared with seven 40 µL drops of activation medium and covered with embryo-tested mineral oil.

3. Transfection of CRISPR-Cas9 into SFFs

  1. (Conditions) Perform all cell culture procedures in a biosafety cabinet or laminar flow hood. Culture SFFs in a T-25 flask with cell culture medium at 37 °C in 5% CO2, 5% O2, and 90% N2 until approximately 70%–80% confluent prior to transfection.
  2. Remove all media from the flask and wash the cells once with 5 mL of Dulbecco’s phosphate-buffered saline (DPBS). Incubate with 1 mL of trypsin-like dissociation enzyme for 5–10 min until the cells dissociate.
  3. Dilute the trypsin-like dissociation enzyme using an equal volume of cell culture medium and centrifuge the cells at 200 × g for 5 min.
  4. (Timing) Prepare the gRNA: Cas9 ribonucleoprotein (RNP) complex by incubating 2.5 µL of 100 µM gRNA with 2.5 µL of 5 µg/µL Cas9 protein for 10 min at room temperature.
    NOTE: Cas9 RNP: gRNA ratios may require optimization depending on the cell type, target gene, and number of targets.
  5. Prepare the nucleofection solution by combining 82 µL of nucleofection solution with 18 µL of supplement solution.
  6. Combine the nucleofection solution with the RNP complex and if using an ssODN, add 2 µL of 200 µM ssODN, then gently mix by pipetting.
  7. After centrifugation, carefully discard the supernatant and place the tube upside down on a lint-free tissue to remove residual medium.
    NOTE: Residual culture medium may affect transfection efficiency and cell viability. Ensure that excess liquid is completely removed before resuspending the cell pellet in the nucleofection solution.
  8. (Critical) Resuspend fibroblasts in the nucleofection solution, then transfer the mixture into a 100 µL nucleocuvette, avoiding bubble formation. Gently tap the nucleocuvette on a hard surface to get the mixture to the bottom of the cuvette.
    NOTE: Bubbles inside the nucleocuvette may interfere with the electroporation.
  9. (Relevant setting) Electroporate the cells using program EH-100, or according to the manufacturer’s recommendations. Allow the cells to recover for 10 min at room temperature.
  10. Transfer the electroporated cells using a transfer pipette into 5 mL pre-equilibrated cell culture medium supplemented with HDR enhancer at a final concentration of 1–2 µM, which may require optimization in other cell types.
  11. (Conditions) Culture the cells in cell culture medium at 37 °C in 5% CO2, 5% O2, and 90% N2 for 48 h prior to genomic DNA isolation.
  12. Remove the medium from the cells, wash once with DPBS, and add 1 mL of trypsin-like dissociation enzyme.
  13. After the cells detach, add 1 mL of cell culture medium to dilute the trypsin-like dissociation enzyme.
  14. Allocate approximately two-thirds of the cell suspension for cryopreservation and one-third for genomic DNA extraction into labeled 1.5 mL tubes. Centrifuge all tubes at 200 × g for 5 min.
  15. Resuspend the cell pellet designated for cryopreservation in 500 µL of freezing medium. Transfer the resuspended cells into cryovials and place them in a controlled-rate freezing container at -80 °C, minimizing the time cells remain in the freezing medium before transfer to -80 °C.
  16. Resuspend the cell pellet designated for DNA extraction in 200 µL of DPBS. Extract genomic DNA using a genomic DNA purification kit according to the manufacturer’s instructions.

4. Analysis of genome editing efficiency

  1. Amplify the target loci from the isolated DNA using high-fidelity DNA polymerase and the previously designed PCR primers.
  2. Confirm PCR amplification by agarose gel electrophoresis in a 1% agarose gel with a 1 kb DNA ladder.
  3. Purify the PCR products using a PCR purification kit and submit for Sanger sequencing.
  4. If a KO was performed, use TIDE software (https://apps.datacurators.nl/tide) to assess editing efficiency36. Use high-quality chromatograms with minimal background signal for accurate editing efficiency analysis.
    1. Open TIDE and name the sample in the Title plot box. Under Guide sequence, input the sequence of the gRNA used.
    2. Under Control Sample Chromatogram, click Browse and upload the chromatogram of the unedited control sample.
    3. Under Test Sample Chromatogram, click Browse and upload the chromatogram of the edited test sample.
    4. Navigate to the Decomposition tab to view the indel spectrum and mutation efficiency. Check the R2 value in the upper-right-hand corner of the indel spectrum.
    5. If the R2 value is less than 0.9, check the Advanced settings box under Parameters and adjust the sliders for the alignment window, decomposition window, and indel size until the R2 value is greater than 0.9 and proper alignment is achieved.
  5. If a KI was performed, use TIDER software (https://apps.datacurators.nl/tider) to assess editing efficiency37.
    1. Open TIDER. Name the sample in the Title plot box. Under Guide sequence, input the sequence of the gRNA used.
    2. Under Control Sample Chromatogram, click Browse and upload the chromatogram of the unedited control sample.
    3. Under Reference Chromatogram, click Browse and upload a chromatogram of a reference sample carrying the desired edit.
      NOTE: If no such sequence exists, one may be generated by PCR amplification using extra primers designed to introduce the mutation into a sequence of DNA38.
    4. Under Test Sample Chromatogram, click Browse and upload the chromatogram of the edited test sample.
    5. Navigate to the Decomposition tab to view the indel spectrum, mutation efficiency, and HDR efficiency. Check the R2 value in the upper-right-hand corner of the indel spectrum.
    6. If the R2 value is less than 0.9, check the Advanced settings box under Parameters and adjust the sliders for the alignment window, decomposition window, distance upstream from break site, and indel size until the R2 value is greater than 0.9 and proper alignment is achieved.
      NOTE: (Critical) A new sequencing may be required if the file sequencing quality is poor.

5. Isolation and screening of single-cell colonies

  1. (Conditions) Thaw and culture transfected SFFs in a T-25 flask overnight at 37 °C with 5% CO2, 5% O2, and 90% N2, or until the cells reach approximately 80% confluence.
  2. Remove the media, wash the cells once with 5 mL of DPBS, and dissociate the cells with 1 mL of trypsin-like dissociation enzyme.
  3. Add cell culture medium to dilute the trypsin-like dissociation enzyme and transfer the cell suspension to a 15 mL conical tube. Centrifuge the cells at 200 × g for 5 min.
  4. Discard the supernatant and resuspend the cell pellet in 1 mL of cell culture medium. Mix gently but thoroughly to obtain a homogeneous single-cell suspension.
  5. (Critical) Prepare a 1:10 dilution of the cell suspension, then count the cells using a hemocytometer with Trypan blue.
  6. Calculate the volume of diluted cell suspension required to seed approximately one cell per well.
    NOTE: For five 96-well plates, prepare approximately 500 cells.
  7. Prepare 10 mL of cell culture medium per 96-well plate in a 50 mL conical tube.
  8. Add the calculated volume of the 1:10 diluted cell suspension to the culture medium and mix well by gentle pipetting and inversion.
  9. Pour the diluted cell suspension into a sterile reagent reservoir.
  10. Dispense 100 µL of the cell suspension into each well of a 96-well plate.
  11. (Conditions) Incubate the plates at 37 °C in 5% CO2, 5% O2, and 90% N2 for 5 days.
  12. Examine each well under a microscope and mark wells containing one colony. Exclude wells containing more than one colony.
    NOTE: Single-cell-derived colonies are usually round, compact, and morphologically homogeneous.
  13. Return the plates to the incubator and culture for an additional 2 days under the same conditions.
  14. After 7 total days of culture in 96-well plates, remove the medium from wells containing a single growing colony. Wash each selected well once with DPBS.
  15. Add 100 µL of trypsin-like dissociation enzyme to each selected well and incubate until the cells detach. Add 100 µL of cell culture medium to each well to dilute the trypsin-like dissociation enzyme.
  16. Transfer the entire volume from each well into one well of a 24-well plate.
  17. Wash the original 96-well plate with additional cell culture medium and transfer the wash to the corresponding 24-well plate well to maximize cell recovery. Bring the final volume in each 24-well plate well to 500 µL with cell culture medium.
  18. (Conditions) Incubate the 24-well plates at 37 °C in 5% CO2, 5% O2, and 90% N2 for 3 days.
  19. Examine the wells under a microscope and select colonies near confluence.
  20. Remove the medium from wells selected for collection, wash once with DPBS, and add 500 µL of trypsin-like dissociation enzyme.
  21. After the cells detach, add 1 mL of cell culture medium to dilute trypsin-like dissociation enzyme.
  22. Freeze cells and isolate genomic DNA according to steps 3.14–3.16.
  23. Amplify the target loci by PCR and confirm the genotype by Sanger sequencing.
  24. Identify wild-type, KO, and KI colonies based on sequencing results. Use TIDE/TIDER analysis as described in steps 4.4 and 4.5 to estimate HDR rates and indel spectrums for validation of correct editing and identification of non-single-cell-derived colonies.
    NOTE: Off-target analysis may also be performed at this step (Supplementary File 1).
  25. Expand only colonies with the desired genotype and normal cellular morphology for downstream SCNT experiments.

6. Ovine oocyte collection and in vitro maturation

  1. Collect sheep ovaries from a local abattoir and transport to the laboratory in 0.9% saline at 25 °C within 4 h of collection.
  2. Mature oocytes in vitro.
    1. Release cumulus-oocyte complexes (COCs) from ovaries using a scalpel blade by slicing the ovarian surface while it’s submerged in oocyte collection medium.
    2. (Timing) Select COCs with at least 1–3 layers of compact cumulus cells and uniform cytoplasm for IVM. Wash COCs in fresh oocyte collection medium and culture in pre-equilibrated IVM medium, with up to 40 COCs per well of 500 µL of IVM medium in a 4-well dish and incubate at 38.5 °C with 5% CO2 for 21–22 h.
  3. Check the maturation of the oocytes.
    1. Move the desired number of COCs using a 200 µL pipette into a 1.5 mL microcentrifuge tube, adding the same volume (1:1) of 0.6 mg/mL hyaluronidase into the tube.
    2. Pipette the mixed solution up and down until all the cumulus cells have been removed. Move the oocytes into a 1 mL HSOF drop and wash the oocytes through three additional HSOF drops.
    3. Using a mouth pipette or a 10 µL pipette and tip, roll and select the oocytes presenting a polar body under a stereomicroscope. The acceptable polar body extrusion rates are 50%–80%.

7. Somatic cell nuclear transfer

  1. Prepare donor cells before SCNT.
    1. Thaw cryopreserved cells 3–5 days prior to SCNT in a water bath at 37 °C for 1–2 min until no ice remains in the tube.
    2. Remove the cell suspension and add an equal volume of cell culture medium into a 15 mL centrifuge tube and centrifuge at 406 x g for 5 min.
    3. Remove the supernatant and resuspend the cell pellet in 500 µL of cell culture medium.
    4. (Conditions) Seed the cells in various concentrations in a 4-well dish in a final volume of 500 µL per well and culture at 37 °C with 5% CO2, 5% O2, and 90% N2.
    5. Replace the cell culture medium every 48 h. After the cells reach full confluency, replace with cell starvation medium 24 h–48h before SCNT.
      ​NOTE: Cell starvation over 48 h may increase the proportion of apoptosis39,40.
  2. Prepare donor cells (day of SCNT).
    1. Remove the starvation medium from the well and wash one time with 500 µL of DPBS (-).
    2. Add 250 µL of trypsin-like dissociation enzyme and incubate at 37 °C for 5 min, or until the cells detach from the dish.
    3. Add 500 µL of cell culture medium to dilute the trypsin-like dissociation enzyme.
    4. Move the cells to a 15 mL centrifuge tube and centrifuge at 406 x g for 5 min.
    5. Remove the supernatant and resuspend the cells in 60–80 µL of HSOF medium and keep at 4 °C until use.
  3. Enucleate the oocytes.
    1. Prepare holding pipettes by pulling thin-wall glass capillaries with a micropipette puller and shaping with a microforge to achieve an inner diameter of 30 µm and an outer diameter of 100 µm.
      NOTE: Both pipettes can also be purchased from commercial suppliers.
    2. Prepare injection pipettes by pulling thin-wall glass capillaries with a micropipette, beveling at a 45° angle with a microgrinder, and shaping with a microforge to achieve an inner diameter of 20–25 µm. The parameters for the micropipette puller, microforge, and microgrinder can be found in a previous report41.
    3. Move the metaphase II (MII)-staged oocytes that presented a polar body, into the drop of enucleation medium. Immobilize the oocyte using a holding pipette while the polar body is in the 4–5 o’clock position.
      ​NOTE: Standard operating procedures for micromanipulation can be referred to the previous report41.
    4. (Critical) Use a beveled injection pipette with a sharp tip to pierce the zona pellucida and aspirate the polar body and 10%–20% of the cytoplasm under the polar body.
    5. Push the removed cytoplasm into the drop and release the oocyte from the holding pipette.
    6. Repeat steps 7.3.2–7.3.4 for all the oocytes until all have been enucleated. Return enucleated oocytes to IVM medium and incubate at 38.5 °C and 5% CO2 for 30 min.
  4. Inject donor cells into enucleated oocytes.
    1. Add the prepared cell suspension into the bottom of the injection medium drop and move enucleated oocytes to the same drop.
    2. Select 1–10 round, healthy cells and aspirate them into the injection pipette.
    3. Use the holding pipette to immobilize an oocyte and use the injection pipette to pierce into the zona pellucida and put one cell between the zona and the ooplasm.
    4. Repeat steps 7.4.2–7.4.3 for all enucleated oocytes and move them to 2–3 mL of HSOF medium in a 35 mm dish before cell fusion.
  5. Fuse enucleated oocytes and donor cells.
    1. Connect the electrofusion pulse generator cables to the wires of the microslide chamber (0.5 mm) and place the chamber onto the microscope stage.
    2. Add 600 µL of fusion medium to the chamber.
    3. (Critical/Relevant setting) Align 4–6 cytoplasm-cell pairs between the two wires of the chamber with the donor cell located at the 6 or 12 o’clock position. Apply two DC electric fusion pulses of 2.0 kV/cm for 40 µs.
    4. Remove the cytoplasm-cell pairs from the fusion chamber and keep them in a new 35 mm dish containing 2–3 mL of HSOF medium.
    5. (Conditions) Repeat steps 7.5.3–7.5.4 until all injected oocytes have undergone the fusion process. Wash the couplets in a post-fusion dish and incubate at 38.5 °C with 5% CO2, 5% O2, and 90% N2 for 30 min. Replace the fusion medium in the chamber every 15–20 min.
  6. Activate and culture embryos.
    1. (Conditions) 30 min after fusion, all fused embryos are first incubated in CB medium at 38.5 °C with 5% CO2, 5% O2, and 90% N2 for 45 min.
    2. Afterward, couplets are activated in 5 µM ionomycin diluted in HSOF for 5 min, followed by incubation in activation medium for 4 h.
      NOTE: Ionomycin medium is freshly prepared before use. Activation at 24–26 h post-maturation is recommended to improve full-term development.
    3. (Conditions) After activation, the reconstructed embryos are washed and cultured in IVC medium, with a maximum of 40 embryos per 40 µL drop at 38.5 °C with 5% CO2, 5% O2, and 90% N2 overnight or until embryo transfer.

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Results

The major steps of the protocol are summarized in Figure 1. We used CRISPR-Cas9 and SCNT to develop a sheep model for cystic fibrosis (CF) containing either a KO indel mutation produced through NHEJ, or the F508del mutation through HDR. Parts of these results have been previously published42,43. We designed gRNAs and PCR primers for both strategies to target Exon 11 of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR...

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Discussion

In this procedure, the design of effective primers, gRNAs, and ssODNs is crucial for successful gene editing. Poor binding of these molecules can hinder gene editing and identification of desirable colonies, inhibiting progression to SCNT. It is recommended to purchase multiple variants of each sequence for testing and optimization prior to proceeding with single-cell colony isolation. Transfection of Cas9 plasmids for KO in fibroblasts typically results in suboptimal editing efficiency, and use of Cas9 RNP is recommende...

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Disclosures

The authors employed Grammarly (Grammarly Inc.) and ChatGPT (OpenAI) to improve the language and readability of the manuscript per accepted uses by scientific publishers. These tools were not used to generate original text, research concepts, data, images, or figures in this proposal. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for this content.

Acknowledgements

This work was supported in part by the Utah Agricultural Experiment Station (UTAO1328) and the Cystic Fibrosis Foundation (award POLEJA23PO).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1 kb DNA ladderThermo Fisher ScientificSM0313DNA size marker
15 mL centrifuge tubeThermo Fisher Scientific12-565-269
1.5 mL microcentrifuge tubeThermo Fisher Scientific05-408-129
4D-Nucleofector Core UnitLonzaAAF-1003B
4D-Nucleofector X UnitLonzaAAF-1003X
4-well dishNunc144444
50 mL centrifuge tubeThermo Fisher Scientific12-565-270
6-DMAPSigma-AldrichD2629
24-well plateCorning3524Cell expansion
96-well plateCorning3596Single-cell colony isolation
AgaroseThermo Fisher ScientificBP160-100Agarose gel electrophoresis
Beaker (250 mL)Kimble14000-250
Benchling softwareBenchlinghttps://www.benchling.com/academicSequence alignment and analysis
IVC mediumIVF Bioscience71005
IVM mediumIVF Bioscience71001
BSAGold BioA-421-250
BTX ECMBTXECM-200Equipment (Discontinued)
BTX microslide chamberBTX450103
Cas9 proteinIntegrated DNA TechnologiesCustomCRISPR-Cas9 genome editing
Chlorhexidine Gluconate (2%)Aspen Vet11584915
CHXSigma-AldrichC7698
CRISPOR softwareCRISPORhttps://crispor.gi.ucsc.edu/Off-target prediction software
CryovialThermo Fisher Scientific12-565-163N
Cytochalasin BSigma-AldrichC6762
Dimethyl sulfoxideFisher ScientificBP231-100Cryopreservation reagent
DMEM high glucoseCytivaSH30022Fibroblast culture medium
DNA purification KitThermo Fisher ScientificK0781Whole Blood Genomic DNA purification
DPBS without Ca/MgCytivaSH30256Used for washing cells and tissues
EthanolPharco111000200CSGL
Fetal bovine serumCytivaSH30396Cell culture supplement
ForcepsSkylar66-6112
Genomic DNA purification kitQiagen69504Genomic DNA extraction
Gentamicin solutionGibco15750-060Used in rinsing medium
Guide RNAIntegrated DNA TechnologiesCustomCRISPR guide RNA
HDR enhancerIntegrated DNA Technologies10007921Enhances homology-directed repair
HemocytometerHausser Scientific02-671-10Cell counting
HemostatsRoboz SurgicalRS-7884
Heparin sodium saltSigma-AldrichH3149
Hot-Start DNA PolymerasePromegaM5123PCR amplification
HyaluronidaseSigma-AldrichH3506
IonomycinSigma-AldrichI0634
M199 MediumCytivaSH30253.01
Mesh Screen (40 mesh)Sigma-AldrichS-0770Opening size 380 μm
MicroforgeNarishigeMF-9
MicrogrinderNarishigeEG-4
Micropipette PullerSutter Instrument CoP-87
Mineral OilKitazato/Dibimed96014
Mouth PipetteThermo Fisher Scientific13-678-20DDisposable pasteur pipettes
Mr. Frosty Freezing ContainerThermo Fisher Scientific15-350-50Controlled-rate freezing container
NucleocuvetteLonzaV4XP-3032Electroporation cuvette
P3 Primary Cell 4D-Nucleofector X Kit LLonzaV4XP-3024Includes nucleofection solution, supplement solution, nucleocuvettes, and transfer pipettes
PCR purification kitQiagen28006PCR product purification
Penicillin-streptomycinGibco15070-063Antibiotic supplement
Petri dish (100 mm)Nunc172958
Petri dish (35 mm)Nunc150460
Reagent ReservoirCELLTREAT3054-1003
ScalpelMedBladesMB 2-22
ScissorsRoboz SurgicalRS-6802
Single-stranded oligodeoxynucleotide donor templateIntegrated DNA TechnologiesCustomHDR donor template
Sodium ChlorideThermo Fisher ScientificBP358-212For saline solution
SucroseSigma-AldrichS1888
Surgical TrayApproved Vendor74262
SYBR Safe DNA Gel StainThermo Fisher ScientificS33102DNA gel stain
T-25 FlasksThermo Fisher Scientific156367Cell culture flask
T-75 flaskCorning430641UCell culture flask
Thin wall glass capillaryWorld Precision InstrumentsTW100-6
TIDE/TIDER softwareTIDE/TIDERhttps://tide.nki.nl/Genome editing analysis software
Trypan blueCytivaSV30084
TrypLE ExpressGibco12605-010Trypsin-like dissociation enzyme

References

  1. Chang MCJ, Grieder FB. The continued importance of animals in biomedical research. Lab Anim (NY). 2024;53(11):295-297.
  2. Bhaduri A, et al. Cell stress in cortical organoids impairs molecular subtype specification. Nature. 2020;578(7793):142-148.
  3. Lin Y, et al. Application of CRISPR/Cas9 system in establishing large animal models. Front Cell Dev Biol. 2022;10:919155.
  4. Seok J, et al. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A. 2013;110(9):3507-3512.
  5. Ineichen BV, et al. Analysis of animal-to-human translation shows that only 5% of animal-tested therapeutic interventions obtain regulatory approval for human applications. PLoS Biol. 2024;22(6):e3002667.
  6. Schuh JCL. Trials, tribulations, and trends in tumor modeling in mice. Toxicol Pathol. 2004;32(1):53-66.
  7. Mak IW, Evaniew N, Ghert M. Lost in translation: animal models and clinical trials in cancer treatment. Am J Transl Res. 2014;6(2):114-118.
  8. Domínguez-Oliva A, et al. The importance of animal models in biomedical research: current insights and applications. Animals (Basel). 2023;13(7):1223.
  9. Li XJ, Lai L. A booming field of large animal model research. Zool Res. 2024;45(2):311-313.
  10. Pinnapureddy AR, et al. Large animal models of rare genetic disorders: sheep as phenotypically relevant models of human genetic disease. Orphanet J Rare Dis. 2015;10(1):107.
  11. Polejaeva IA, Rutigliano HM, Wells KD. Livestock in biomedical research: history, current status and future prospective. Reprod Fertil Dev. 2015;28(2):112-124.
  12. Kelly OM, et al. Towards clinical translation of nanomedicines: formulation scale-up and model systems. Adv Drug Deliv Rev. 2026;234:115862.
  13. Jinek M, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.
  14. Pfeiffer P, Vielmetter W. Joining of nonhomologous DNA double strand breaks in vitro. Nucleic Acids Res. 1988;16(3):907-924.
  15. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51:503-512.
  16. Takata M, et al. Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 1998;17(18):5497-5508.
  17. Jiang S, Shen QW. Principles of gene editing techniques and applications in animal husbandry. 3 Biotech. 2019;9(1):28.
  18. Javaid N, Choi S. CRISPR/Cas system and factors affecting its precision and efficiency. Front Cell Dev Biol. 2021;9:761709.
  19. Wang J, et al. Application of gene editing technology in livestock: progress, challenges, and future perspectives. Agriculture (Basel). 2025;15(20):2155.
  20. Saleh-Gohari N, Helleday T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 2004;32(12):3683-3688.
  21. Miyaoka Y, et al. Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep. 2016;6:23549.
  22. Wen W, Zhang XB. CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells. Exp Hematol. 2022;110:13-19.
  23. Schnieke AE, et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997;278(5346):2130-2133.
  24. Tang L, González R, Dobrinski I. Germline modification of domestic animals. Anim Reprod. 2015;12(1):93-104.
  25. Perisse IV, et al. Improvements in gene editing technology boost its applications in livestock. Front Genet. 2021;11:614688.
  26. Kwon DH, Gim GM, Yum SY, Jang G. Current status and future of gene engineering in livestock. BMB Rep. 2024;57(1):50-59.
  27. Mehravar M, Shirazi A, Nazari M, Banan M. Mosaicism in CRISPR/Cas9-mediated genome editing. Dev Biol. 2019;445(2):156-162.
  28. Niu Y, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell. 2014;156(4):836-843.
  29. Yen ST, et al. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol. 2014;393(1):3-9.
  30. Ni W, et al. Efficient gene knockout in goats using CRISPR/Cas9 system. PLoS One. 2014;9(9):e106718.
  31. Polejaeva IA. 25th anniversary of cloning by somatic cell nuclear transfer: generation of genetically engineered livestock using somatic cell nuclear transfer. Reproduction. 2021;162(1):F11-F22.
  32. Hodges CA, Stice SL. Generation of bovine transgenics using somatic cell nuclear transfer. Reprod Biol Endocrinol. 2003;1:81.
  33. Galli C. Current techniques of gene editing in pigs for xenotransplantation. Transpl Int. 2025;38:13807.
  34. Concordet JP, Haeussler M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 2018;46(W1):W242-W245.
  35. Liu Y, et al. A retrospective analysis of sheep generated by somatic cell nuclear transfer. Theriogenology. 2024;227:102-111.
  36. Brinkman EK, Chen T, Amendola M, van Steensel B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 2014;42(22):e168.
  37. Brinkman EK, et al. Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Res. 2018;46(10):e58.
  38. Brinkman EK, Van Steensel B. Rapid quantitative evaluation of CRISPR genome editing by TIDE and TIDER. Methods Mol Biol. 2019;1961:29-44.
  39. Dalman A, et al. Synchronizing cell cycle of goat fibroblasts by serum starvation causes apoptosis. Reprod Domest Anim. 2010;45(5):e46-e53.
  40. Sadeghian-Nodoushan F, et al. Mimosine as well as serum starvation can be used for cell cycle synchronization of sheep granulosa cells. Chin J Biol. 2014;2014(1):851736.
  41. Page RL, Malcuit CM. Micromanipulation techniques for cloning. In: Principles of Cloning. Elsevier; 2014. p. 39-51.
  42. Viotti Perisse I, et al. Sheep models of F508del and G542X cystic fibrosis mutations show cellular responses to human therapeutics. FASEB Bioadv. 2021;3(10):841-854.
  43. Fan Z, et al. A sheep model of cystic fibrosis generated by CRISPR/Cas9 disruption of the CFTR gene. JCI Insight. 2018;3(19).
  44. Berthault C, et al. Highly efficient ex vivo correction of COL7A1 through ribonucleoprotein-based CRISPR/Cas9 and homology-directed repair to treat recessive dystrophic epidermolysis bullosa. J Invest Dermatol. 2024;144(6):1322-1333.e13.
  45. Martufi M, et al. Single-step, high-efficiency CRISPR-Cas9 genome editing in primary human disease-derived fibroblasts. CRISPR J. 2019;2(1):31-40.
  46. Bloomer H, Khirallah J, Li Y, Xu Q. CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells. Adv Drug Deliv Rev. 2022;181:114087.
  47. Blocher R, et al. Effect of laparoscopic ovum pick-up on ovine in vitro oocyte maturation and somatic cell nuclear transfer embryo development. Anim Reprod Sci. 2026;292:108251.
  48. Campbell KH, Loi P, Otaegui PJ, Wilmut I. Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev Reprod. 1996;1(1):40-46.
  49. Chen Y, Niu Y, Ji W. Genome editing in nonhuman primates: approach to generating human disease models. J Intern Med. 2016;280(3):246-251.
  50. Liu D, Cao D, Han R. Recent advances in therapeutic gene-editing technologies. Mol Ther. 2025;33(6):2619-2644.
  51. Campbell K, Alberio R. Reprogramming the genome: role of the cell cycle. Biosci Proc. 2019.
  52. Chia G, et al. Genomic instability during reprogramming by nuclear transfer is DNA replication dependent. Nat Cell Biol. 2017;19(4):282-291.
  53. Wang X, et al. Epigenetic reprogramming during somatic cell nuclear transfer: recent progress and future directions. Front Genet. 2020;11:205.
  54. Loi P, Iuso D, Czernik M, Ogura A. A new, dynamic era for somatic cell nuclear transfer? Trends Biotechnol. 2016;34(10):791-797.
  55. Zhao J, et al. Histone deacetylase inhibitors improve in vitro and in vivo developmental competence of somatic cell nuclear transfer porcine embryos. Cell Reprogram. 2010;12(1):75-83.
  56. Huang J, et al. BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei. Reproduction. 2016;151(1):39-49.
  57. Han C, et al. Overexpression of TET3 in donor cells enhances goat somatic cell nuclear transfer efficiency. FEBS J. 2018;285(14):2708-2723.
  58. Wang S, et al. Application of gene editing technology in resistance breeding of livestock. Life (Basel). 2022;12(7):1070.
  59. Houdebine LM. Production of pharmaceutical proteins by transgenic animals. Comp Immunol Microbiol Infect Dis. 2009;32(2):107-121.

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