Sex chromosome–targeted Cas9 knock-in and functional validation in chicken primordial germ cells

Abstract

Sex-specific control of genome editing remains a significant challenge in birds. Chickens exhibit a ZW sex-determination system in which the Z and W chromosomes encode genes essential for sex differentiation and germline development, providing a rationale for sex-linked genome engineering. In this study, we established the sex chromosome–linked knock-in system for Cas9 in chicken primordial germ cells (PGCs). Donor constructs carrying Cas9-GFP were engineered to integrate into either the Z chromosome (DMRT1–DMRT3 intergenic region) or the W chromosome (5’ region of HINTW locus). The targeting strategy was validated in DF-1 fibroblasts and PGCs, where site-specific integration was confirmed by junction PCR and sequencing. Functionality of the integrated Cas9 was verified by targeting two different loci, demonstrating efficient genome cleavage at NHEJ1 loci and indel-associated loss of GFP fluorescence following GFP targeting. The knock-in PGCs expressed Cas9 protein while retaining germ cell markers and migration capacity, demonstrating preservation of germline identity. Collectively, our findings establish a sex chromosome–linked Cas9 knock-in system in chicken PGCs and demonstrate that these sites support stable Cas9 expression without compromising germline characteristics, thereby providing a practical foundation for controlled, sex-specific genome engineering in avian research.

Introduction

Precise and controlled genome editing in avian species is essential for both basic and applied research. In chickens, primordial germ cells (PGCs) provide a practical platform for generating genome edited chicken lines, as they can be expanded in vitro while maintaining their progenitor characteristics and subsequently contribute to the germline following transplantation into recipient embryos (van de Lavoir, et al., 2006; Choi, et al., 2010; Macdonald, et al., 2010; Song, et al., 2014). Although CRISPR/Cas9 technology has significantly improved genome editing in avian systems (Idoko-Akoh, et al., 2018; Lee, et al., 2020; Atsuta, et al., 2022; Zhou, et al., 2025), stable and sex chromosome-linked Cas9 expression within the germline has not been fully established, limiting the development of genetic models requiring predictable sex-linked inheritance or sustained genome editing capacity during development. Targeted genomic integration represents an alternative approach to address the limitations associated with random insertion and unintended mutagenesis (Yamamoto and Gerbi, 2018; Chen, et al., 2024). In chickens, CRISPR/Cas9- nonhomologous end joining (NHEJ) strategy has enabled targeted insertion in PGCs, supporting efficient genome editing and germline transmission (Lee, et al., 2019; Rengaraj, et al., 2022). Several autosomal and sex-chromosomal candidate insertion sites, such as cROSA, GAPDH, and regions on the Z and W chromosomes, have been characterized for stable transgene expression in DF-1 cells (Dehdilani, et al., 2023; Wu, et al., 2024; Jung, et al., 2026). However, sex-linked regions in PGCs that support stable transgene expression without compromising germ cell identity or developmental competence have yet to be established.

Sex chromosomes represent biologically relevant targets for genome editing in birds, as they play key roles in sex differentiation and germline development. Unlike mammals, avian species possess a ZZ/ZW sex-determination system in which males are homogametic (ZZ) and females are heterogametic (ZW) (Fridolfsson, et al., 1998; McQueen, et al., 2001). The Z chromosome contains regulators of sex differentiation, including DMRT1, a Z-linked gene required for male sex determination (Smith, et al., 2009a; Lee, et al., 2021). The intergenic region between DMRT1 and DMRT3 constitutes a defined non-coding interval that allows targeted insertion while avoiding direct disruption of coding sequences and has been utilized in previous studies for Z-linked reporter integration (Kang, et al., 2023). In contrast, the W chromosome is largely heterochromatic but harbors female-specific loci such as HINTW, which is expressed during early embryonic development and serves as a W-linked genetic marker (Ceplitis and Ellegren, 2004; Smith, et al., 2009b; Ayers, et al., 2013; Nagai, et al., 2014). Although these regions are biologically well characterized, their potential as defined genomic safe harbors for functional genome-editing components remains to be explored.

In this study, we developed a CRISPR/Cas9–NHEJ-mediated knock-in strategy to integrate a Cas9-GFP cassette into the Z chromosome (DMRT1–DMRT3 intergenic locus) and the W chromosome (HINTW region) in chicken PGCs. And we further investigated whether these loci could sustain stable Cas9 expression while preserving germ cell identity and germline competence. Collectively, this work establishes a sex chromosome–linked Cas9 integration strategy and broadens the genetic toolkit for sex-linked genome editing in avian species.

Materials and methods

Animal ethics statement

All procedures involving fertilized chicken eggs were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Missouri (Protocol No. 42644).

Construction of plasmids

Previously established CRISPR/Cas9 all-in-one plasmids were employed to target the DMRT1–DMRT3 intergenic region, the HINTW locus, and to generate the corresponding donor constructs (Lee, et al., 2017). gRNA sequences were designed using CRISPOR (Haeussler, et al., 2016), which ranks candidates by predicted on-target activity and assesses potential off-target interactions based on PAM-proximal mismatch sensitivity. For cloning gRNAs into the PX459 vector, complementary oligonucleotides were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). The oligos were annealed using a stepwise temperature-reduction program (95°C for 30 s, 72°C for 2 min, 37°C for 2 min, and 25°C for 2 min), after which the duplexes were inserted into PX459 via Golden Gate assembly. All assembled plasmids were confirmed by Sanger sequencing. Insertion of the Cas9–GFP–puromycin cassette into either the DMRT1–DMRT3 intergenic region on the Z chromosome or the HINTW locus on the W chromosome was carried out using the CAG-Cas9-T2A-EGFP-IRES-Puro construct (Addgene #78311; RRID:Addgene_78311). For experiments requiring Cas9-independent gRNA expression (GFP or NHEJ1 targeting), annealed oligonucleotides were cloned into a custom backbone containing the U6 promoter and gRNA scaffold but lacking the Cas9 coding sequence. All oligonucleotide sequences used in this study are provided in Supplementary Table 1.

Chicken cell culture

Chicken DF-1 fibroblast cells (ATCC® CRL-12203, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, Cytiva, Marlborough, MA, USA) and 1 × antibiotic–antimycotic solution (Gibco). Cultures were kept at 37°C with 5% CO₂ in a humidified incubator and passaged routinely using TrypLE™ Express (Gibco).

Chicken PGCs were isolated from the circulating blood of Pure Columbian embryos at Hamburger and Hamilton (HH) stage 13-16 following a previously described protocol with minor adjustments (Whyte, et al., 2015). Approximately 1–2 μL of embryonic blood was collected from the dorsal aorta and immediately diluted in 300 μL FAcs medium. FAcs medium consisted of Ca²⁺-free DMEM (Gibco) supplemented with 100 μM CaCl₂, 1 × B-27 (Gibco), 2 mM GlutaMAX (Gibco), 1 × non-essential amino acids (Gibco), 55 μM β-mercaptoethanol (Gibco), 1.2 mM sodium pyruvate (Gibco), 0.2% chicken serum (Biowest), 0.2% ovalbumin (Sigma-Aldrich), 0.2% sodium heparin (Sigma-Aldrich), 10 μg/mL ovotransferrin (Sigma), 25 ng/mL Activin A (R&D Systems), and 4 ng/mL FGF2 (R&D Systems). PGCs were kept in low-adherence culture at 37°C and 5% CO₂ during early expansion and subsequently transitioned to standard feeder-free PGC medium for long-term maintenance.

Cell transfection

To validate the CRISPR/Cas9 constructs, DF-1 cells and PGCs were transfected with plasmids targeting either the Z or W chromosomes. DF-1 cells were seeded in 12-well plates at 3 × 10⁵ cells per well to reach ∼70% confluence at the time of transfection. For each well, 4 µg of CRISPR/Cas9 plasmid DNA was diluted in Opti-MEM (Thermo Fisher Scientific) and mixed with Lipofectamine 2000 (Thermo Fisher Scientific) at a 1:1 (w/w) DNA-to-reagent ratio. After a 5-min incubation at room temperature, the transfection mixture was added dropwise to the cells. The medium was replaced with fresh DF-1 growth medium 24 h later. For PGC transfection, 2 µg of CRISPR/Cas9 plasmid DNA was mixed with Lipofectamine 2000 in 1 mL Opti-MEM and applied to 2 × 10⁵ PGCs in suspension. After 4 h, the transfection mixture was replaced with standard PGC culture medium.

For targeted gene insertion, DF-1 cells and PGCs were co-transfected with donor plasmids containing the gRNA target site and a puromycin-selectable marker together with the CRISPR/Cas9 plasmids. The same transfection procedure described above was used, with a total of 10 µg plasmid DNA per well (3.3 µg of each plasmid). Cells were allowed to recover in complete medium for 24 h, followed by puromycin selection (Thermo Fisher Scientific) to prevent the overgrowth of untransfected WT cells. Cells were maintained under puromycin selection until the cell populations uniformly expressed GFP. Targeted integration was subsequently evaluated in the selected cell populations.

To assess Cas9 activity in knock-in PGCs, cells were transfected with 2 µg of a gRNA-expressing plasmid targeting GFP or NHEJ1, following the same protocol as for knockout experiments. After 24 h, cells were subjected to dual selection with puromycin (1 µg/mL) and neomycin/G418 (300 µg/mL) to enrich for successfully modified populations.

Flow cytometry

Flow cytometric analysis was performed using WT, Cas9–GFP knock-in, and GFP knockout Z-Cas9 PGCs and W-Cas9 PGCs. The cells were dissociated and resuspended in phosphate-buffered saline (PBS), and fluorescence signals were analyzed on a Cytek Aurora flow cytometer (Cytek Biosciences, Fremont, CA, USA). Data were processed using FlowJo software (Treestar, Ashland, OR, USA).

T7E1 assay and genomic DNA sequencing

Genomic DNA was isolated from transfected DF-1 cells, PGCs, or knock-in PGCs to evaluate genome editing efficiency. Target loci were amplified by PCR using locus-specific primers (Supplementary Table 1). The PCR products were denatured and reannealed to form heteroduplexes and then digested with 5 U of T7 endonuclease I (New England Biolabs, Ipswich, MA, USA) at 37°C for 20 min. The digestion products were separated on a 1.5% agarose gel. Densitometric analysis of T7E1 assay bands was performed using ImageJ software. The fraction cleaved was calculated by dividing the combined intensity of the cleavage products by the total intensity of the cleavage products and uncleaved band. The estimated indel frequency was calculated using the formula: % indel = 100 × (1 − √(1 − fraction cleaved)). For sequence verification, PCR amplicons were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and subjected to Sanger sequencing. Sequence data were analyzed using Geneious Prime software (Biomatters Ltd., Auckland, New Zealand).

Knock-in junction PCR

Genomic DNA was isolated from knock-in PGCs using a DNA extraction kit (Zymo Research, Irvine, CA, USA). To verify correct integration of the donor construct, PCR was performed using primer pairs specific to the 5′ and 3′ junctions of the target locus. Both forward and reverse integration events were examined separately, resulting in a total of four knock-in-specific PCR reactions (primer sequences are listed in Supplementary Table 1). Genomic DNA was amplified using DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Representative amplicons were purified and confirmed by Sanger sequencing.

Analysis of off-target mutations in knock-in PGCs

Potential off-target sites were identified using the CRISPOR online prediction tool, and the three highest-scoring candidate sites were selected for each gRNA. These predicted sites shared 16 of 20 nucleotides with the gRNA sequence (four mismatches), including stretches of up to 12 consecutive matched nucleotides (Supplementary Fig. 2A). Genomic DNA isolated from knock-in PGCs was used as a template for PCR amplification of the corresponding off-target regions. The resulting amplicons were cloned into the pGEM-T Easy vector and subjected to Sanger sequencing. Sequence analysis was performed using Geneious Prime software.

Western blot

Proteins were extracted from WT or knock-in PGCs using RIPA buffer (Thermo Fisher Scientific) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Approximately 10 µg of total protein was separated on 4–20% gradient SDS–PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto iBlot 2 PVDF membranes (Thermo Fisher Scientific). Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: anti-Cas9 (MA1-202; Thermo Fisher Scientific) or anti-β-tubulin (A01410; GenScript, Piscataway, NJ, USA). After washing, membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Thermo Fisher Scientific), imaged with an iBright 1500 Imaging System, and analyzed using iBright Analysis Software (Thermo Fisher Scientific).

RT-PCR

Total RNA was extracted from WT and knock-in PGCs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Total RNA samples were then reverse transcribed into cDNAs using the SuperScript III Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cDNAs were amplified by PCR using specific primer sets of germ cell-specific genes, along with GAPDH (Supplementary Table 1).

Migration assay

Z- or W-chromosome–targeted Cas9–GFP knock-in PGCs were collected by centrifugation at 200 × g for 4 min and resuspended in Hanks’ balanced salt solution (HBSS). Approximately 1,000 cells were injected into the dorsal aorta of White Leghorn embryos at HH stages 13–16. The injection sites were sealed with Parafilm™ (Bemis, Neenah, WI, USA), and the eggs were incubated further until HH stage 28. Fluorescent cells that migrated to the gonadal region were visualized under a fluorescence microscope.

Statistical analysis

Statistical analysis was performed using Prism software (GraphPad, Boston, MA). One-way ANOVA was used to compare the means of each group with the control group (WT), and P < 0.05 was considered statistically significant.

Results

CRISPR/Cas9-mediated targeting of chicken sex chromosomes in DF-1 cells and PGCs

To evaluate the activity of gRNAs targeting the chicken sex chromosomes, a gRNA was designed for the intergenic region between DMRT1 and DMRT3 on the Z chromosome (Fig. 1A). CRISPR/Cas9 constructs were introduced into DF-1 cells, and genomic DNA extracted from the cells was analyzed. T7E1 assays detected cleavage products at the target site, indicating induction of double-strand breaks at the intended locus (Fig. 1B). PCR amplicons spanning the target site were cloned into a TA vector and subjected to Sanger sequencing, which confirmed indel mutations at the predicted cleavage sites (Fig. 1C). Introduction of the CRISPR/Cas9 constructs into PGCs resulted in indel mutations at the target loci (Figs. 1D and E). T7E1 band densitometry estimated indel frequencies of 27% and 24% in DF-1 cells and PGCs, respectively. Next, a gRNA targeting the 5′ region of HINTW on the W chromosome was designed (Fig. 1F). T7E1 assays and sequencing analysis confirmed indel mutations at the target loci in both CRISPR/Cas9-transfected DF-1 cells and PGCs (Figs. 1G–J). T7E1 band densitometry estimated indel frequencies of 39% and 37% in DF-1 cells and PGCs, respectively. These results validate the activity of the selected Z- and W-specific gRNAs for targeted genome editing in chicken cells.

Fig. 1.

Validation of CRISPR/Cas9 gRNAs targeting the sex chromosomes in chicken DF-1 and PGCs. (A) Schematic diagram of the intergenic region between DMRT1 and DMRT3 loci on the Z chromosome, indicating CRISPR/Cas9 gRNA target site (orange bar). (B-C) Validation of Z-chromosome targeting gRNA in DF-1 cells. (B) T7E1 assay and (C) Sanger sequencing confirmed efficient cleavage at the target sites following transfection with CRISPR/Cas9 plasmids. (D-E) Validation of Z-chromosome targeting gRNA in PGCs. (F) Schematic diagram of the HINTW gene on the W chromosome, indicating CRISPR/Cas9 gRNA target site (orange bar). (G-H) Validation of W-chromosome targeting gRNA in DF-1 cells. (G) T7E1 assay and (H) Sanger sequencing confirmed activity of the W-specific gRNA. (I-J). Validation of W-chromosome targeting gRNA in PGCs. In sequencing panels, gRNA target sequences are shown in orange, PAMs in light blue, deleted bases are indicated by strikethroughs, and insertions by lowercase letters. Estimated indel frequencies were calculated from T7E1 band densitometry and are indicated in the corresponding panels.

Targeted knock-in of Cas9-GFP into the Z chromosome in chicken PGCs

Targeted knock-in strategy was first evaluated in DF-1 cells prior to its application in PGCs. For CRISPR/Cas9-non-homologous end joining (NHEJ)–mediated targeted gene insertion into the Z or W chromosome, DF-1 cells were co-transfected with a donor plasmid carrying the Cas9–2A–GFP cassette and puromycin resistance gene, a CRISPR/Cas9 plasmid targeting the Z or W chromosome, and a CRISPR/Cas9 plasmid targeting the donor plasmid (Fig. 2A). GFP fluorescence was detected in the transfected DF-1 cells after drug selection, indicating successful integration of the Cas9–2A–GFP cassette. In contrast, non-transfected WT DF-1 cells showed no detectable fluorescence under identical imaging conditions (Supplementary Fig. S1A). Targeted integration was confirmed by junction PCR spanning both the 5′ and 3′ insertion sites (Supplementary Fig. S1B).

Fig. 2.

Targeted knock-in of Cas9-GFP into the Z chromosome of chicken PGCs. (A) Schematic representation of the CRISPR/Cas9-mediated targeting strategy for the intergenic region between DMRT1 and DMRT3 on the chicken Z chromosome. The donor plasmid for this site carries a CAG promoter driving Cas9 expression, followed by a 2A peptide sequence, GFP (green fluorescent protein; G), an internal ribosome entry site (IRES; I), puromycin-resistance (shown as P), and an ampicillin-resistance gene (shown as A) for bacterial selection. The entire transgene is flanked by homology arms corresponding to the target locus to enable NHEJ-mediated integration. (B) Representative bright-field and fluorescence images of knock-in PGCs expressing GFP. Non-transfected WT PGCs show no fluorescence under identical imaging conditions. PGCs transfected with the knock-in vector targeting the intergenic region between DMRT1 and DMRT3 on the Z chromosome exhibited strong GFP signals following puromycin selection, confirming reporter gene expression. Scale bar, 50 µm. (C) Junction PCR confirming targeted integration of the donor construct into the DMRT1–DMRT3 locus. (D) Sanger sequencing of PCR products verified integration of the donor sequence at both 5′ and 3′ junctions, with characteristic indels generated by non-homologous end joining (NHEJ). Both forward and reverse integration orientations were identified. (E) Western blot analysis of Cas9 protein expression in Z chromosome–targeted Cas9-GFP knock-in PGCs using anti-Cas9 antibody. α-tubulin served as the loading control.

The validated knock-in strategy was then applied to chicken PGCs. Knock-in vectors were co-transfected into PGCs, and following puromycin selection, stable GFP-expressing PGC lines were established (Fig. 2B). Targeted integration at the DMRT1–DMRT3 intergenic locus was confirmed by junction PCR spanning both the 5′ and 3′ insertion sites (Fig. 2C). Sanger sequencing of the junction amplicons verified donor integration at the expected genomic location, with indels consistent with NHEJ-mediated repair, and both forward and reverse insertion orientations were detected (Fig. 2D). Western blot analysis confirmed Cas9 protein expression in Z-Cas9 knock-in PGCs, whereas no Cas9 signal was observed in WT PGCs (Fig. 2E).

Generation of W-linked Cas9-GFP knock-in PGCs

The W chromosome–targeting knock-in vector system was first validated in DF-1 cells (Supplementary Fig. S1) and subsequently applied to target the HINTW locus on the W chromosome in chicken PGCs (Fig. 3A). Following co-transfection of knock-in vectors and puromycin selection, stable GFP-expressing PGC lines were established (Fig. 3B). Targeted integration at the HINTW locus was confirmed by junction PCR spanning both the 5′ and 3′ insertion sites (Fig. 3C). Sanger sequencing verified donor integration at the expected genomic location with indels consistent with NHEJ-mediated repair, and both forward and reverse insertion orientations were identified (Fig. 3D). Western blot analysis confirmed Cas9 protein expression in W-Cas9 knock-in PGCs, while no Cas9 signal was detected in WT cells (Fig. 3E).

Fig. 3.

Targeted knock-in of Cas9-GFP into the W chromosome of chicken PGCs. (A) Schematic illustration of the targeting strategy for the HINTW locus on the chicken W chromosome. The donor plasmid for this site carries a CAG promoter driving Cas9 expression, followed by a 2A peptide sequence, GFP (green fluorescent protein; G), an internal ribosome entry site (IRES; I), puromycin-resistance (P), and an ampicillin-resistance gene (A) for bacterial selection. The entire transgene is flanked by homology arms corresponding to the target locus to enable NHEJ-mediated integration. (B) Representative bright-field and fluorescence images of W-linked knock-in PGCs expressing GFP following puromycin selection. Non-transfected WT PGCs show no fluorescence under identical imaging conditions. Scale bars, 50 µm. (C) Junction PCR confirming targeted integration of the donor construct into the HINTW locus on the W chromosome. (D) Sanger sequencing of junction PCR products verified correct donor integration with characteristic indels introduced by non-homologous end joining (NHEJ). Both forward and reverse insertion orientations were detected. (E) Western blot analysis of Cas9 protein expression in W-linked Cas9-GFP knock-in PGCs using anti-Cas9 antibody. α-tubulin served as loading control.

Evaluation of predicted off-target sites in Z- and W-linked knock-in PGCs

To assess potential off-target mutations of the Z- and W-targeting gRNAs, the three highest-ranked candidate loci for each gRNA were selected for analysis (Supplementary Fig. 2A). Genomic DNA from Z- and W-linked knock-in PGCs was amplified across each locus, and the PCR products were subjected to Sanger sequencing. No insertions or deletions were detected at any of the examined sites (Supplementary Fig. 2B), suggesting that editing was confined to the intended target regions under the conditions tested.

Functional validation of Cas9 activity in sex chromosome–linked knock-in PGCs

To determine whether the integrated Cas9 was functionally active, Z- and W-linked knock-in PGCs were transfected with a gRNA targeting the integrated GFP sequence as an exogenous target (Fig. 4A). Transfection of the GFP-targeting gRNA resulted in a marked reduction in GFP-positive cells in both Z- and W-linked knock-in PGCs. Flow cytometric analysis confirmed a substantial decrease in GFP fluorescence compared with knock-in PGCs without gRNA transfection (Fig. 4B), consistent with Cas9-mediated cleavage of the integrated GFP sequence. T7E1 assays further demonstrated indel formation at the GFP target site (Fig. 4C), and Sanger sequencing verified mutation frequencies of 100% (6/6) in Z-linked knock-in PGCs and 85.7% (6/7) in W-linked knock-in PGCs (Fig. 4D).

Fig. 4.

Functional validation of Cas9 activity in sex chromosome–targeted knock-in PGCs. (A–D) Assessment of Cas9 function by targeting the integrated GFP reporter gene. (A) Schematic of the Cas9–GFP knock-in cassette showing the GFP-specific gRNA target site (orange bar). (B) GFP fluorescence and flow cytometric analysis of sex chromosome–targeted knock-in PGCs following transfection with a GFP-targeting gRNA expression plasmid. GFP fluorescence images show a clear reduction in GFP. Flow cytometric analysis further confirms a marked decrease in GFP-positive cells, indicating Cas9-mediated disruption of the integrated GFP sequence. Scale bar, 100 μm. (C) T7E1 assay demonstrating indel formation at the GFP target site in knock-in PGCs transfected with the GFP-gRNA construct. (D) Sanger sequencing of the targeted GFP region confirmed indel mutations consistent with Cas9-mediated genome cleavage. (E–G) Validation of Cas9 activity at an endogenous locus (NHEJ1). (E) Diagram of the NHEJ1 gene with the gRNA target site indicated (orange bar). (F) T7E1 assay showing indel formation at the NHEJ1 locus in knock-in PGCs transfected with the NHEJ1-gRNA plasmid. (G) Sanger sequencing analysis of the edited NHEJ1 locus in both Z- and W-chromosome knock-in PGCs. In sequencing panels, gRNA sequences are shown in orange, PAMs in light blue, deleted bases are marked by strikethroughs, and insertions by lowercase letters.

To further evaluate genome editing activity at an endogenous locus, knock-in PGCs were transfected with a gRNA targeting the endogenous NHEJ1 gene (Fig. 4E). Indel formation at the NHEJ1 locus was detected by T7E1 analysis in both Z- and W-linked knock-in PGCs (Fig. 4F), and Sanger sequencing confirmed highly efficient indel formation at the expected cleavage site (Fig. 4G). Together, these findings demonstrate that integrated Cas9 at either the Z or W locus retains robust genome-editing activity, enabling efficient modification of both exogenous and endogenous targets in chicken PGCs.

Germline competence of Z- and W-linked Cas9 knock-in PGCs

RT-PCR analysis confirmed expression of the germ cell–specific genes, specifically CVH (chicken vasa homolog) and DAZL (deleted in azoospermia-like), in Z- and W-linked Cas9 knock-in PGCs, comparable to WT PGCs, suggesting that targeted integration did not compromise germ cell characteristics (Figs. 5A and B). To further evaluate germline competence, Z- or W-linked knock-in PGCs were microinjected into recipient embryos. GFP-positive donor cells successfully colonized the embryonic gonads, indicating that the Z- and W-linked knock-in PGCs retained germline competence (Fig. 5C).

Fig. 5.

Germline competence of Z- and W-linked Cas9–GFP knock-in PGCs. (A-B) RT-PCR analysis of germ cell–specific markers (CVH and DAZL) in Z- and W-linked knock-in PGCs. GAPDH was used as an internal control. (C) Migration capacity of Z- and W-chromosome knock-in PGCs. Approximately 1,000 PGCs were microinjected into recipient embryos at HH stages 13–16, and migrated cells were detected in the gonadal region at HH stages 28–30. Scale bar, 100 μm.

Discussion

This study demonstrates that defined loci on the chicken Z and W chromosomes can support CRISPR/Cas9–NHEJ–mediated targeted knock-in of a large transgene cassette in PGCs without compromising germline properties. While previous avian genome editing efforts have largely focused on autosomal insertion sites (Mukae, et al., 2020; Shi, et al., 2020; Ichikawa, et al., 2021; Wang, et al., 2023), our data extend this framework to the sex chromosomes and show that these regions are compatible with stable Cas9 expression and functional genome editing in germline-competent cells. This expands the range of genomic sites available for targeted integration in chickens and establishes sex chromosome–linked engineering as a feasible strategy.

In chicken PGCs, homology-directed repair (HDR)–mediated DNA cassette replacement has been reported, but its efficiency can vary depending on donor design and experimental conditions (Dimitrov, et al., 2016). In contrast, CRISPR/Cas9–nonhomologous end joining (NHEJ)–mediated targeted knock-in provides a practical alternative that does not rely on long homology arms and can facilitate efficient insertion at predefined cleavage sites (Auer, et al., 2014). In the present study, a large Cas9–GFP cassette was successfully integrated into both Z- and W-linked loci, and integration occurred in both forward and reverse orientations as expected for NHEJ-mediated repair. Because the expression cassette was driven by a ubiquitous CAG promoter, insertion orientation did not influence Cas9 expression or genome-editing activity. These findings indicate that, under the experimental conditions tested, NHEJ-mediated targeted integration represents an effective and functionally reliable strategy for large transgene insertion in avian germ cells.

An important consideration in sex chromosome–targeted genome editing is whether the selected loci can accommodate large transgene insertion without perturbing germ cell identity or local genomic integrity. The DMRT1–DMRT3 intergenic region on the Z chromosome and the HINTW locus on the W chromosome were selected as candidate sex chromosome–linked integration sites to avoid direct disruption of protein-coding sequences, rather than for manipulation of their endogenous gene functions. Despite the proximity of the Z-linked target site to genes involved in sex differentiation and the potential presence of local regulatory elements, integration of the Cas9–GFP cassette did not alter the expression of core germ cell markers such as CVH and DAZL. Interestingly, the super-enhancer (SE) histone markers H3K4me1 and H3K27ac are highly enriched within the DMRT1–DMRT3 intergenic region in the developing mouse forebrain (Kubota, et al., 2018), suggesting that conserved SEs may exist in the syntenic region of chicken cells. Because literature regarding SEs in this specific chicken genomic region is currently lacking, future study will involve mining epigenetic profiles from web-based databases like FAANG (Andersson, et al., 2015) or the Chicken GTEx Atlas (Guan, et al., 2025), followed by functional validation of these putative SEs using genome editing. Similarly, stable Cas9 expression and functional editing were observed from the W-linked HINTW locus, even though the avian W chromosome is largely heterochromatic. These results indicate that both loci are functionally permissive for large transgene integration and remain transcriptionally competent in PGCs. Although comprehensive transcriptomic profiling would be required to formally designate these regions as genomic safe harbors, our data provide functional evidence that they behave as genomically tolerant integration sites within the PGC context.

Preservation of germline competence is a critical requirement for PGC-based genome editing strategy (Han, et al., 2015). In this study, both Z- and W-linked knock-in PGCs migrated to recipient embryonic gonads following transplantation, indicating retention of germline potential. Nevertheless, confirmation of stable germline transmission will require analysis of G1 progeny and assessment of transmission frequency. In addition, further studies will be needed to evaluate the stability of transgene expression across generations.

Cas9, expressed from the targeted sex chromosome loci, retained robust genome editing activity in PGCs. Efficient editing was observed at both an exogenous reporter sequence and an endogenous genomic target, demonstrating that locus-integrated Cas9 can mediate double-strand breaks at independent sites in PGCs. The establishment of stable Cas9 knock-in PGC lines reduces the need for repeated Cas9 vector delivery, which can be inefficient or associated with cellular stress in avian germ cells, and may facilitate sequential or multiplex genome-editing applications. The long-term cellular impact of constitutive Cas9 expression from a genomic insertion site was not examined in detail in this study and will require additional evaluation.

From a broader perspective, positioning genome editing components on sex chromosomes introduces predictable inheritance patterns within the avian ZZ/ZW system. Z-linked integration is expected to follow male-biased transmission, whereas W-linked integration is restricted to females. Although the present study does not directly implement sex-sorting or production strategies, the ability to localize functional genome-editing machinery to specific sex chromosomes establishes a conceptual framework for future development of sex-dependent genetic models and breeding applications in poultry. In parallel, no insertions or deletions were detected at the highest-ranked predicted off-target sites in the edited PGC lines. While these findings support the target specificity of the selected gRNAs under the experimental conditions tested, further analyses at the genome-wide level will be necessary to comprehensively evaluate genomic integrity in established lines, particularly in the context of sustained Cas9 expression.

Taken together, this study demonstrates that the chicken Z and W chromosomes can serve as effective platforms for CRISPR/Cas9-NHEJ–mediated targeted knock-in in PGCs. By demonstrating functional Cas9 activity together with preservation of germ cell properties, this study extends avian genome engineering beyond autosomal loci and provides a practical foundation for sex chromosome–linked genome engineering strategies in poultry.

CRediT authorship contribution statement

Kyung Min Jung: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. Sabrina Islam Mony: Writing – original draft, Validation, Investigation. Paula R Chen: Writing – review & editing, Resources. Kiho Lee: Writing – review & editing, Resources. Hong Jo Lee: Validation, Supervision, Methodology, Conceptualization.

Disclosures

The authors have no conflicts of interest to declare.