All animal experimental protocols in this study were approved and carried out in strict accordance with guidelines for the Animal Care and Use Committee at Academy of Military Medical Sciences (approval number IACUC-DWZX-2023–301). The study strictly adhered to the Guidelines for the Care and Use of Laboratory Animals issued by the National Institutes of Health.

Three sgRNAs targeting the goat FGF5 locus were selected in this study. PX458 plasmids, containing Cas9 and U6-sgRNA co-expression backbones, were obtained from Addgene and linearized using BbsI-HF (NEB, United States). Complementary sgRNA oligos were synthesized, annealed, and individually ligated into the linearized PX458 backbone vector to construct PX458-sgRNA plasmids. Plasmids were purified using the Endofree Plasmid Maxi Kit (Qiagen, Germany).

A PX458-sgRNA plasmid targeting the pig RAG1 gene was constructed using the method described above. Subsequently, the EGFP reporter gene in the PX458 backbone was replaced with EBFP to generate the PX458-EBFP-sgRNA plasmid.

Forward and reverse donor templates for HAs-CMV-mCherry-polyA, HAs-EF1α-EGFP-polyA, and HAs-CMV-FLAG-hBChE-polyA were constructed using the In-Fusion Snap Assembly Master Mix (Takara, Japan). For forward templates, left and right homology arms (approximately 1 kb each) were combined within the 5′and 3′ends of CMV-mCherry-polyA, EF1α-EGFP-polyA, or CMV-FLAG-hBChE-polyA, respectively. Conversely, reverse templates were assembled by combining these homology arms with the 3′and 5′ends of the respective constructs. The hBChE gene was obtained by extracting RNA from 293T cells using the MiniBEST Universal RNA Extraction Kit (Takara, Japan), followed by cDNA synthesis with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Japan). The complete open reading frame (ORF) regions of hBChE cDNA was amplified using PrimeSTAR GXL Premix Fast, Dye plus (Takara, Japan). The HAs-P2A-mCherry-TFB donor templates were also constructed using the In-Fusion Snap Assembly Master Mix (Takara, Japan). All donor templates were ligated into the linearized vectors using the In-Fusion Snap Assembly Master Mix (Takara, Japan). Plasmids were purified with the Endofree Plasmid Maxi Kit (Qiagen, Germany), and donor DNA fragments were amplified using PrimeSTAR GXL Premix Fast, Dye plus (Takara, Japan).

Goat fibroblast cells (gFCs) were cultured in DMEM/F12 (Gibco, United States) supplemented with 10% fetal bovine serum (FBS, Gibco, United States) and 1% penicillin/streptomycin (Gibco, United States) at 37 °C in a 5% CO₂ atmosphere. Pig fetal fibroblast cells (PFFs) were cultured in MSC medium (ScienCell, United States) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco, United States) at 38.5 °C in a 5% CO₂ atmosphere. Upon reaching approximately 80%–90% confluency, the gFCs and PFFs were trypsinized for subsequent passage or cryopreservation.

For transfection, 2.5 × 10⁶ cells were mixed with 10 μg PX458-sgRNA plasmid and 5 μg of donor template in 100 μL of nucleofector solution. Transfection was performed using the Amaxa Basic Nucleofector Kit for Primary Fibroblasts and the Nucleofector 2b Device (Lonza, Switzerland) with program A-033. Post-transfection, cells were transferred to 6-well plates for further culture.

To evaluate HDR efficiency, PX458-sgRNA plasmids and HAs-P2A-mCherry-TFB donor DNA fragment were co-transfected into gFCs. After 48 h of cultivation, cells were stained with the Zombie NIR Fixable Viability Kit (BioLegend, United States) and resuspended in PBS containing 3% FBS. mCherry-positive (mCherry⁺) viable cells were quantified using a BD FACSAria II flow cytometer.

For evaluating both forward and reverse knock-in efficiencies, PX458-sgRNA plasmids were co-transfected into gFCs with either forward or reverse HAs-CMV-mCherry-polyA donor templates. After 48 h, cells were stained and resuspended in complete DMEM/F12 medium with 20% FBS and 1% penicillin/streptomycin. 2000 viable cells expressing both double EGFP and mCherry were then sorted into each well of a 6-well plate using a BD FACSAria II flow cytometer and cultured for 12 days. mCherry⁺ viable cells were quantified using a BD FACSAria II flow cytometer.

Similarly, PX458-EBFP-sgRNA plasmids were co-transfected into PFFs with either forward or reverse HAs-EF1α-EGFP-polyA donor templates. After 48 h, cells were stained with the 7-AAD and resuspended in complete MSC medium with 20% FBS and 1% penicillin/streptomycin. 5,000 viable cells expressing both double EBFP and EGFP were then sorted into each well of a 6-well plate using a BD FACSAria II flow cytometer and cultured for 14 days. EGFP-positive (EGFP ⁺) viable cells were quantified using a BD FACSAria II flow cytometer.

PX458-EBFP-sgRNA plasmids were transfected into PFFs. Single EBFP-positive (EBFP⁺) cells were sorted into 96-well plates using a BD FACSAria II flow cytometer and cultured for 12 days to allow colony formation. Individual cell colonies were lysed in a buffer containing 4% Tris-HCl (1 M, pH = 8.0), 0.9% TritonX-100, 0.9% NP-40, and 0.4 mg/mL proteinase K (all from Solarbio, China). Lysis was performed at 65 °C for 30 min, followed by heating at 95 °C for 15 min, and cooling to 4 °C. Lysates were directly used for PCR identification with PrimeSTAR GXL Premix Fast, Dye plus (Takara, Japan). Primers are listed in Supplementary Table S1. PCR products were subsequently cloned into the pEASY-Blunt Zero Cloning Vector (TransGen, China) and verified by Sanger sequencing.

To establish hBChE knock-in cell lines, PX458-sgRNA plasmids were co-transfected into gFCs with either forward or reverse HAs-CMV-FLAG-hBChE-polyA donor templates. Single EGFP⁺ were sorted into 96-well plates using a BD FACSAria II flow cytometer and cultured for 12 days to allow colony formation. Cell clones were lysed using the method described above, and the resulting lysates were used directly as templates for PCR amplification. Primers P1 and P2 detected forward knock-in hBChE clones; N1 and N2 detected reverse knock-in hBChE clones. Primer PN determined zygosity. All primers are listed in Supplementary Table S2. PCR products were analyzed on a 1% agarose gel and visualized using a ChemiDoc imaging system (Bio-Rad, United States). Positive PCR products were subsequently cloned into the pEASY-Blunt zero-cloning vector (TransGen, China) and confirmed by Sanger sequencing.

Total RNA was extracted using TRIzol reagent (Mei5bio, China) and subsequently reverse-transcribed into cDNA using the PrimeScript RT Kit with gDNA Eraser (Takara, Japan). rhBChE mRNA expression was quantified using primer Q1 and TB Green Premix Ex Taq II (Takara, Japan) on a CFX96 Real-Time System (Bio-Rad, United States), with β-Actin serving as the reference gene. Relative expression levels were analyzed using the 2^-ΔΔCt method. Primer sequences in this study are listed in Supplementary Table S3.

hBChE-positive (hBChE+) and wild-type (WT) gFCs were cultured in 24-well plates, fixed with 4% paraformaldehyde tissue fixation solution (Biosharp, BL539A, China) for 15 min, permeabilized with 0.1% Triton X-100 (diluted from 10% sterile stock in PBS; Beyotime, ST797, China) for 10 min, and blocked with 5% bovine serum albumin blocking buffer (Solarbio, SW3015, China) for 60 min. Cells were incubated overnight at 4 °C with Anti-Butyrylcholinesterase antibody (Abcam, ab236577, United Kingdom) diluted 1:1,000 in universal antibody diluent (NCM Biotech, WB500D, China), followed by an incubation with CoraLite488-Conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, China), diluted at a ratio of 1:1,000 for 60 min in the dark. Nuclei were stained with DAPI solution (Solarbio, C0065, China) for 10 min. Visualization was performed using an Olympus IX71 microscope.

Total proteins were extracted from gFCs and goat skin tissue using the Minute Total Protein Extraction Kit for Animal Cultured Cells/Tissues (Inventbiotech, United States). Protein concentrations were then determined with Pierce BCA Protein Assay Kits (Thermo Fisher Scientific, United States). Equal amounts of protein samples were separated on 4%–12% SDS-PAGE gels (Huaxingbio, China) and transferred to PVDF membranes (Thermo Fisher Scientific, United States). Membranes were blocked with 5% Difco skim milk (BD Biosciences, United States) for 60 min at room temperature. For rhBChE detection, membranes were incubated overnight at 4 °C with Recombinant Anti-Butyrylcholinesterase antibody (1:1,000; Abcam, ab151554, United Kingdom) and HRP-conjugated GAPDH antibody (1:10,000; Proteintech, China) as a loading control. After washing, HRP-conjugated Goat Anti-Rabbit IgG (H + L) (1:5,000; Proteintech, China) was applied for 60 min at room temperature to detect rhBChE. For FLAG-tagged protein detection, membranes were incubated overnight at 4 °C with HRP Anti-DDDDK tag antibody (1:1,000; Abcam, ab49763, United Kingdom) and HRP-conjugated Beta Actin antibody (1:5,000; Proteintech, China). Protein bands were detected using an ECL chemiluminescence detection system (Thermo Fisher Scientific, United States) and visualized with a ChemiDoc imaging system (Bio-Rad, United States).

WT gFCs were stained using the Zombie NIR Fixable Viability Kit (BioLegend, United States), and 3,000 viable cells were sorted into each well of a 96-well plate using a BD FACSAria II flow cytometer. These cells were cultured in DMEM/F12 complete medium (Gibco, United States) with varying concentrations of organophosphorus pesticides (OPPs, Aladdin, United States) for 24 h at 37 °C in a 5% CO₂ incubator. Cell viability was assessed using the CCK-8 Cell Proliferation and Activity Detection Kit (Mei5bio, China), and the median lethal dose (LD₅₀) of OPPs was determined.

Subsequently, both WT gFCs and hBChE⁺ gFCs were stained and sorted as above, then cultured in medium containing OPPs at the LD₅₀ concentration. After 24 h, cell morphology was observed under an Olympus IX71 microscope, and viability was compared using the CCK-8 assay.

To evaluate potential off-target effects of the CRISPR-Cas9 system in vivo, candidate off-target loci were identified using Cas-OFFinder. Selected loci were amplified by PCR and subcloned into pEASY-Blunt zero-cloning vector (TransGen, China) for validation through Sanger sequencing. Primer sequences are listed in Supplementary Table S4.

Mature oocytes were collected from healthy female donor laoshan dairy goats and cultured in TCM199 medium (Sigma-Aldrich, United States) supplemented with 2% FBS (Gibco, United States). The oocytes were incubated in TCM199 medium (Sigma-Aldrich, United States) supplemented 5 μg/mL cytochalasin B (CB, Sigma-Aldrich, United States) and 5 μg/mL Hoechst 33,342 (Beyotime, China) for 10 min, followed by enucleation using micromanipulation techniques. Subsequently, hBChE⁺ gFCs were injected into the enucleated oocytes and fused using the ECM2001 Electrocell Manipulator (BTX Inc, United States). The embryos were cultured in TCM199 medium containing 10 μg/mL Cycloheximide (Sigma-Aldrich, United States) and 5 μg/mL CB for an additional 5 h, then transferred to developmental medium for further cultivation and embryo transfer tests. Two months after embryo transfer, pregnancy rates in recipient goats were assessed by ultrasonography.

The data were analyzed using Student’s t-test and One-way ANOVA. Results are presented as mean ± standard deviation (SD). Statistical significance was determined as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

The limited HDR efficiency poses a significant challenge for the establishment of genome editing animal models. In this study, we evaluated the HDR efficiency of three sgRNAs targeting the goat FGF5 locus to identify the one with the highest HDR rate. Three types of homology arms (HAs)-linked P2A-mCherry-TFB fluorescent reporter donor templates were constructed, each targeting one of the three sgRNA sites (Figure 1A). The “TFB” sequence, consisting of T2A, 3X FLAG, and the hBChE gene, was included to extend the total length of the donor template, ensuring comparable sizes between the P2A-mCherry-TFB and CMV-FLAG-hBChE-polyA constructs. The HAs-P2A-mCherry-TFB system enabled the fusion of FGF5 and mCherry through the P2A translational skipping peptide, allowing the expression of mCherry under the control of the endogenous promoter of FGF5. For the knock-in experiments, transfection with PX458-sgRNA plasmids alone served as the negative control. As an experimental control, PX458-sgRNA plasmids were co-transfected with the HAs-P2A-mCherry-TFB donor DNA fragment into gFCs. After 48 h of cultivation, flow cytometry analysis revealed that the proportion of mCherry⁺ viable cells in the sgRNA3 group reached 0.5%, significantly higher than in the other groups (Figures 1B,C). Therefore, sgRNA3 exhibited the highest HDR efficiency among the three candidates.

In CRISPR/Cas9-mediated genome editing, targeted knock-in of an expression vector at a specific genomic locus can be performed either in the forward orientation, aligning with the transcriptional direction of the target gene (from 5′to 3′), or alternatively in the reverse orientation, inserting the expression vector from the 3′to 5′direction relative to the target gene’s orientation. However, few studies have explored the reverse knock-in method.

To evaluate the efficiency of forward versus reverse knock-in methods, we selected three sgRNAs targeting the FGF5 locus and constructed corresponding forward and reverse donor templates (HAs-CMV-mCherry-polyA) (Figure 2A). In the forward knock-in groups, PX458-sgRNA plasmids were co-transfected with forward donor templates into gFCs, while in the reverse knock-in groups, PX458-sgRNA plasmids were co-transfected with reverse donor templates. PX458-sgRNA plasmids co-transfected with CMV-mCherry-polyA fragments lacing homology arms served as negative controls. 48 h after transfection, 2000 double-positive (EGFP and mCherry⁺) cells were sorted from each group and cultured for 12 days to eliminate transient expression and allow degradation of non-integrated donor templates (Figure 2B). Flow cytometry analysis revealed that, for all three sgRNAs, the proportion of mCherry⁺ cells was significantly higher in the reverse knock-in groups compared to the forward knock-in groups (Figures 2C,D). Among them, sgRNA3 exhibited the highest HDR efficiency in both orientations. These results demonstrate that the reverse knock-in method achieves higher HDR efficiency than the forward knock-in method across all tested target sites within the FGF5 locus.

To further explore whether this reverse donor orientation strategy is broadly applicable across different genomic loci and livestock species, we extended our investigation to the pig RAG1 gene. A specific sgRNA was designed, and the corresponding PX458-sgRNA plasmid was constructed. Subsequently, the EGFP gene in the PX458-sgRNA plasmid was replaced with the EBFP blue fluorescent reporter to construct the RAG1-targeting plasmid PX458-EBFP-sgRNA (Figure 3A). This plasmid was transfected into Wuzhishan PFFs to assess knockout efficiency. All 10 examined clones exhibited mutations in the RAG1 locus (Figure 3B), confirming efficient CRISPR/Cas9-mediated editing and validating the suitability of this site for targeted integration. Based on this result, we constructed corresponding forward and reverse donor templates (HAs-EF1α-EGFP-polyA) (Figure 3C). The PX458-EBFP-sgRNA plasmid was co-transfected with either the forward or reverse donor template into PFFs. PX458-EBFP-sgRNA plasmid co-transfected with EF1α-EGFP-polyA fragments lacing homology arms served as negative controls. Cells were transfected with the donor template alone as a blank control. In each well of a 6-well plate, 5000 EBFP and EGFP double-positive cells were sorted. After 8 days of culture, EGFP⁺ monoclonal colonies were observed under a fluorescence microscope (Figure 3D). Following 14 days of culture, flow cytometric analysis revealed that the HDR efficiency was significantly higher in the reverse-integration groups compared to the forward-integration groups (Figures 3E,F).

These results demonstrate that reverse donor orientation enables superior knock-in efficiency not only at a different genomic locus but also in a different species.

To generate goat fibroblast cell clones with either forward or reverse knock-in of the hBChE gene, we constructed forward and reverse donor templates (HAs-CMV-FLAG-hBChE-polyA) based on sgRNA3 targeting the FGF5 locus (Figure 4A). PX458-sgRNA plasmids were co-transfected with either the forward or reverse donor templates into gFCs. After transfection. EGFP⁺ single cells were isolated via flow cytometry and cultured for 12 days.

A total of 190 cell clones derived from forward knock-in were screened by PCR using P1, P2, and PN primers, resulting in the identification of 2 hBChE⁺ clones (positive rate: 1.05%) (Figure 4B). Among these, 1 clone was homozygous and 1 clone was heterozygous (each accounting for 0.53%) (Figure 4D). Meanwhile, 245 cell clones derived from reverse knock-in were screened using N1, N2, and PN primers, yielding 8 hBChE⁺ clones (positive rate: 3.27%) (Figure 4C). Of these, 3 clones were homozygous clones (1.22%) and 5 clones were heterozygous (2.04%) (Figure 4D). All PCR products were validated by Sanger sequencing to confirm precise genome integration (Figure 4E). Taken together, these results demonstrate that the reverse knock-in method generated a greater number of hBChE⁺ cell clones compared to the forward knock-in method.

The rhBChE expression at the mRNA level was detected in 4 cell lines: one reverse knock-in homozygous line (R1), one reverse knock-in heterozygous line (R2), one forward knock-in homozygous line (F1), and one forward knock-in heterozygous line (F2). Quantitative real-time PCR analysis demonstrated that the expression levels of rhBChE mRNA in the reverse knock-in cell lines (R1 and R2) were significantly higher than those in the forward knock-in cell lines (F1 and F2) (Figure 5A) At the protein level, rhBChE expression was analyzed by immunofluorescence and western blotting. Due to the poor viability of the F1 cell line, only R1, R2 and F2 were assessed for protein expression. Immunofluorescence staining revealed enhanced rhBChE protein expression in all 3 cell lines (Figure 5E). Western blotting analysis demonstrated that rhBChE protein levels were significantly elevated in R1, R2 and F2 compared to WT gFCs, consistent with the mRNA expression results (Figures 5B,C). To specifically detect the rhBChE and avoid potential interference from endogenous BChE in gFCs, an antibody targeting the FLAG tag was used. Western blotting confirmed robust expression of FLAG-tagged rhBChE in R1-positive cells (Figure 5D).

To evaluate the resistance of these cell lines to OPPs, the LD₅₀ for OPPs in WT gFCs was determined to be 154.9 μM (Figure 5F). Subsequently, four cell lines (R1, R2, F2 and WT) were individually cultured in complete medium supplemented with OPPs at 154.9 μM for 24 h. Observation under a microscope showed with OPPs that the hBChE⁺ cell lines maintained their activity and showed no significant alterations in cell morphology (Figure 5H). Furthermore, according to the results of the CCK-8 assay, the hBChE⁺ cell lines demonstrated significantly enhanced resistance to OPPs compared to WT gFCs (Figure 5G).

To check the potential off-target effects in the hBChE⁺ cell lines, we identified a total of 10 potential off-target sites using Cas-OFFinder software. These sites were amplified by PCR and subjected to Sanger sequencing. The sequencing results were compared with the corresponding NCBI reference sequences. No nucleotide mutations were observed at any of the predicted off-target loci, indicating the absence of off-target effects in the edited cells (Figure 6A).

Given the superior cell viability and rhBChE expression in the reverse knock-in hBChE cell lines, we selected a reverse knock-in homozygous clone (R1) for somatic cell nuclear transfer (SCNT) to produce cloned goats. A total of 104 MII-stage oocytes were collected from 10 superovulated Laoshan dairy goats, of which 92 (88.46%) were deemed suitable for SCNT. Following electrofusion, 66 reconstructed embryos were obtained and subsequently were transferred into 4 female recipients. Pregnancy diagnosis confirmed that one recipient became pregnant, and a goat was born. Genomic DNA extracted from the blood of the goat was subjected to PCR using N1 and N2 primers, confirming the integration of the hBChE gene (Figure 6B). Sanger sequencing further validated the precise integration of the hBChE gene into the cloned goat genome (Figure 6C). To assess rhBChE protein expression in the cloned goat, total protein was extracted from the skin tissue and analyzed by western blotting. The results demonstrated high-level expression of the rhBChE protein in the cloned goat (Figure 6D).