Research note: Generation of ovalbumin-null chickens and characterization of altered protein compositions in their egg whites

TL;DR

Scientists created chickens without ovalbumin, a major egg white protein, and found that other proteins increased to compensate.

Contribution

Successfully generated homozygous ovalbumin-null chickens using CRISPR/Cas9, overcoming prior developmental limitations.

Findings

OVAL-null chickens were successfully generated through mating heterozygous individuals.

Embryos developed and hatched from OVAL-deficient eggs, though with a 52.30±14.42% reduced hatching rate.

Other major egg white proteins increased in concentration, suggesting compensatory accumulation.

Abstract

Chickens are considered an efficient bioreactor platform for production of recombinant proteins due to their high egg laying rate and high capacity to produce proteins in egg white. Ovalbumin (OVAL) comprises 54% of egg white proteins, and targeted insertion of a recombinant protein construct into the OVAL locus leads to significant accumulation of the recombinant protein in egg white. However, it was reported that embryos could not develop or hatch from eggs laid by heterozygous OVAL-knockout hens in which OVAL gene was replaced by foreign protein coding sequences, a limitation that restricted the generation of homozygous OVAL-knockout chickens. In this study, we specifically targeted the OVAL locus using CRISPR/Cas9 and successfully generated OVAL-null chickens by mating heterozygous individuals. Both heterozygous and homozygous OVAL knockout embryos developed and hatched, notably, we found that embryos could develop and hatch from OVAL-deficient eggs, although the hatching rate was reduced by 52.30±14.42%. Furthermore, analysis revealed that concentrations of other major egg white proteins increased, indicating a compensatory accumulation of proteins in response to the removal of OVAL. Collectively, this study demonstrates that OVAL-null chicken lines can be established and that these chickens produce eggs with altered egg white protein compositions.

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Introduction

Chickens have been an important food resources for humans, providing high-quality protein through both meat and eggs. Recent advances in genome editing technologies, together with efficient culture system for chicken primordial germ cells (PGCs), have generated synergistic effects that enable the development of high-valued chicken breeds. Representative outcomes of these advances include the development of disease-resistance chickens as well as chickens improved for specific economically important traits (Idoko-Akoh et al., 2023; Kim et al., 2020)

In addition to their value as a protein source, chickens are considered an efficient bioreactor for production of expensive therapeutic proteins because hens lay more than 300 eggs per year and egg white contains abundant proteins. Ovalbumin (OVAL) comprises 54% of egg white proteins, and thus, the OVAL promoter is highly active in oviduct tubular gland cells (Lillico et al., 2005). Therefore, several studies have sought to express therapeutic protein constructs using synthetic OVAL promoters and successful accumulation of functional therapeutic proteins in egg whites has been reported. These results demonstrated that chicken bioreactors could be efficient production platform for functional therapeutic proteins, although the overall yield of accumulated proteins remains relatively low (Lillico et al., 2007). After development of precise genome editing technologies, such as transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), gene editing of ovalbumin (OVAL) gene and precise targeting of therapeutic protein construct into OVAL locus has been successfully performed and targeted therapeutic proteins massively accumulated in egg white (Park et al., 2014; Oishi et al., 2018). Recently, targeted integration of foreign protein construct into liver-specific gene, such as Albumin (ALB), has been reported to enable the accumulation of recombinant protein in the bloodstream and subsequently in egg yolk (Park et al., 2023). In addition to the production of recombinant proteins in eggs, targeted gene editing of the Ovomucoid (OVM) gene, one of the major egg allergens, has also been reported, resulting in genome edited hens that produce OVM-null eggs (Mukae et al., 2021). Collectively, these findings demonstrate that precise gene editing of egg protein-related genes can be effectively applied both to produce valuable recombinant proteins and to modulate egg white protein composition, thereby enabling the generation of high-valued eggs.

However, to our knowledge, there have been no reports demonstrating the successful production of OVAL-null eggs, although gene editing of the OVAL gene has been previously achieved (Park et al., 2014; Oishi et al., 2016). Interestingly, eggs laid by generation 1 (G1) heterozygous OVAL-knockout hens, in which foreign protein construct was successfully targeted in OVAL locus and resultant foreign protein was massively accumulated in egg white, display changes the shape of egg white and decreased egg white volume (Oishi, et al., 2018). Furthermore, embryos could not develop in eggs from heterozygous OVAL-knockout hens, indicating a detrimental effect of the inserted foreign protein on embryos, or that changes in egg white composition hindered embryo development (Oishi, et al., 2018). Also, it could be assumed that OVAL in egg white is essential for development of chicken embryos. However, the exact reason for development failure in eggs from heterozygous OVAL-knockout hen has not been demonstrated.

In this study, gene targeting was performed via CRISPR/Cas9-mediated non-homologous end joining (NHEJ), which enables the homology-independent insertion of linearized donor plasmids (Lee et al., 2019). We specifically targeted OVAL locus in order to suppress the expression and secretion of OVAL protein in egg white, and subsequently analyzed changes in egg white composition after OVAL knockout. Contrary to a previous report, we found that embryos could develop in eggs from heterozygous OVAL knockout chickens, which enabled the generation of homozygous OVAL knockout birds. These OVAL-null chickens laid eggs lacking OVAL and exhibiting altered egg white compositions. Ultimately, we confirmed that embryos could successfully hatch from these OVAL-deficient eggs.

Materials & methods

Experimental animals and animal care

The management and experimental use of chickens were approved by the Institutional Animal Care and Use Committee (IACUC), Seoul National University (SNU-220311-1). The experimental animals were cared according to a standard management program at the University Animal Farm and Institute of Laboratory Animal Resources, Seoul National University.

Construction of CRISPR/Cas9 expression plasmids and donor plasmids

The CRISPR/Cas9 vector targeting intron #1 of chicken OVAL gene was constructed using PX459 vector (Addgene plasmid #62988) provided by Feng Zhang. To insert guide RNA (gRNA) sequences into the CRISPR/Cas9 plasmid, sense and antisense oligonucleotides were designed and synthesized (Bioneer, Daejeon, Korea). These oligonucleotides were annealed under the following thermocycling conditions: 30 sec at 95°C, 2 min at 72°C, 2 min at 37°C, and 2 min at 25°C. For targeted gene insertion into the chicken OVAL gene, a donor cassette containing a part of the intron 1 region, a part of exon 2 including the translation start site of the chicken ovalbumin gene, the human IgG gene, and the CMV promoter and puromycin resistance gene was synthesized in the pBHA vector backbone (Bioneer, Daejeon, Korea).

Production of OVAL targeted chickens

For PGC line establishment, the White Leghorn (WL) male PGCs were maintained and sub-passaged on knockout DMEM (Invitrogen, Carlsbad, CA) supplemented with 20% FBS (Invitrogen), 2% chicken serum (Sigma-Aldrich, St.Louis, MO), 1 × nucleosides (Millipore, Temecula, CA), 2 mM l-glutamine, 1 × nonessential amino acids, β-mercaptoethanol, 10 mM sodium pyruvate, 1 × antibiotic–antimycotic (Invitrogen), and human basic fibroblast growth factor (10 ng/ml; Sigma-Aldrich). Chicken PGCs were cultured in an incubator at 37 °C under an atmosphere of 5% CO_2_ and 60–70% relative humidity. The PGCs were sub-cultured onto mitomycin-inactivated mouse embryonic fibroblasts at 5- to 6-day intervals via gentle pipetting. After transfection of Cas9 expressing and donor vectors, puromycin was added to culture medium for 2 days to select OVAL targeted PGCs. The selected OVAL targeted PGCs were transferred to dorsal aorta of Hamburger & Hamilton (HH) stage 14-17 of Korean Ogye (KO) embryo. After sexual maturation, sperm of the potent recipient KO germline chimera was collected and inseminated to wild type KO hens. The donor WL PGCs derived progenies were distinguished by their feather color and the species of these G1 donor PGC derived progenies were hybrid of White Leghorn and Korean Ogye. Targeted insertion of donor plasmid in PGCs and progenies was validated using primers specific to 5′ and 3′ junctions of insertion site and subsequent sequencing analysis. The genome edited G1 hybrid progenies (wing tag number H1384 and H1385) were interbred to produce G2 genome edited chickens. All the egg-based experiments were conducted using eggs collected from the G2 progenies of 50 weeks after hatching. For sequencing analysis, the amplicons were annealed to the pGEM-T Easy Vector (Promega, WI, USA) and sequenced using an ABI Prism 3730XLDNAAnalyzer (Thermo Fisher Scientific, MA, USA). The sequences were compared against assembled genomes using the Basic Local Alignment Search Tool (BLAST; http://blast.ncbi.nlm.nih.gov).

Reverse transcription polymerase chain reaction (RT-PCR) analysis

Total RNA was extracted from organs using Trizol reagent (15596026, Thermo Fisher Scientific, MA, USA). The complementary DNA (cDNA) was synthesized using Superscript III First-Strand Synthesis System (18080051, Thermo Fisher Scientific, MA, USA). The cDNA was amplified using target gene-specific primer set.

Quantification of proteins in egg white using ELISA

For ELISA analysis, egg white was diluted in phosphate buffered saline (PBS) and analyzed concentration of proteins in egg white using specific ELISA kits. The ELISA kits used in this study were as follows: Chicken Ovalbumin ELISA kit (CSB-E13315C, Cusabio, TX, USA), Chicken Ovotransferrin ELISA kit (ab157694, Abcam), Chicken Lysozyme ELISA kit (CSB-E13182C, Cusabio), Chicken Ovomucin ELISA kit (MBS108958, MyBioSource, CA, USA). The ELISA was performed according to each manufacturer’s instructions.

SDS-PAGE and western blot

For western blot analysis, egg white was diluted in PBS and denatured at 95°C for 5 minutes in equally mixed with 2 × Laemmli sample buffer (BioRad). Then the protein was separated on 10% sodium dodecyl sulfate (SDS) – polyacrylamide gels. The resolved proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane and blocked for 1hr at room temperature. The membrane was incubated with a suitable primary antibody, followed by an appropriate horseradish peroxidase – conjugated secondary antibody (Santa Cruz Biotech, TX, USA). The primary antibodies used in this study were as follows: anti-Ovalbumin (NB600-922, Novus, CO, USA), anti-Ovotransferrin (MBS715799, MyBioSource), anti-Ovomucoid (MBS715888, MyBioSource) anti-Lysozyme (GTX48846, GeneTex, CA, USA) and anti-Avidin (GTX19507, GeneTex). Immunoreactive proteins were visualized using the ECL Select Western Blotting Detection Reagent (GE Healthcare Bio-Science, NJ, USA). Signals were detected using a BioRad ChemiDoc XRS imaging system (BioRad).

Immunohistochemistry

Chicken magnum of oviduct retrieved from OVAL^+/+^and OVAL^-/-^ chickens were paraffin-embedded and sectioned (thickness, 9 μm). After deparaffinization, sections were washed three times with 1 × phosphate-buffered saline (PBS) and blocked with a blocking buffer (5% goat serum and 1% bovine serum albumin in PBS) for 1 hr at room temperature. Sections were then incubated at 4°C overnight with an anti-OVA primary antibody (NB600-922, Novus). After washing three times with PBS, sections were incubated with fluorescence-conjugated secondary antibodies (Alexa Fluor 594, Invitrogen, MA, USA) for 1 h at room temperature. After washing three times with PBS, sections were mounted with Prolong Gold antifade reagent with DAPI and imaged using fluorescence microscope.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (GraphPad Software, CA, USA). Significant differences between groups were determined by a one-way ANOVA analysis of variance with Bonferroni’s multiple comparison test and the unpaired t-test. A value of P < 0.05 indicated statistical significance.

Results and discussion

To achieve targeted gene insertion specifically into the OVAL locus, a Cas9 expression vector targeting intron 1 and a donor plasmid containing the human IgG construct were introduced into PGCs via the CRISPR/Cas9-NHEJ-mediated knock-in method. (Fig. 1A) (Lee, et al., 2019). After co-transfection of the Cas9 expression vector and donor plasmid into WL PGCs, we performed puromycin selection and established an *OVAL-*targeted PGC line. Targeted insertion of the donor plasmid in PGCs was confirmed by specific genomic DNA PCR analysis at the 5′ and 3′ junctions of the target site (Fig. 1B). To produce OVAL knockout chickens, we transplanted the WL (I/I) PGCs into the dorsal aorta of KO (i/i) recipient embryos at HH stage 14–17. After sexual maturation, the germline chimeric KO roosters (G0) were test-crossed with wild-type KO hens, and donor-derived hybrid (G1, I/i) progenies were successfully hatched (Fig. 1C). Production of OVAL-targeted progenies was validated by genomic DNA amplification at the 5′ and 3′ junctions of the target site and subsequent sequencing analysis (Fig. 1D). After production of G1 heterozygous OVAL knockout chickens (OVAL^±^), we performed mating between G1 OVAL^±^individuals after sexual maturation, and G2 progenies were hatched (Fig. 1E). Hatched G2 progenies were genotyped by performing genomic DNA PCR analysis with specific primers for the wild type OVAL allele and modified OVAL allele and found that wild type (OVAL^+/+^), OVAL^±^ and OVAL^-/-^ G2 progenies were hatched (Fig. 1F). These results indicate that reduction of OVAL expression by one-half does not have detrimental effects on embryos and OVAL^-/-^ chickens can be produced by mating between OVAL^±^chickens. Previous studies reported that embryos cannot develop normally in eggs laid by OVAL^±^chickens, and that the shape of egg white in eggs of OVAL^±^chickens is visibly changed (Oishi, et al., 2018). The common features of previous studies reporting impaired embryogenesis in eggs laid by OVAL^±^chickens are the high-level accumulation of exogenous proteins in egg white and altered morphology of egg white. Therefore, the type and quantity of recombinant protein accumulating in egg white and resultant changes of the egg white composition may have detrimental effects on embryo development.Fig. 1Production of OVAL-null chickens. (A) Schematic illustration of OVAL targeting donor plasmid containing OVAL intron #1 and exon 2, human IgG construct and CMV Puro^R^ expression cassette. The donor plasmid will be inserted into gRNA target site mediated by non-homologous end joining (NHEJ) DNA repair process. PAM sequence is indicated as blue bar and gRNA recognition site is indicated as red bar. PCR primers specific for 5′ and 3′ junctions are indicated as arrows. (B) Validation of targeted insertion of donor plasmid into target site in PGCs by genomic DNA PCR of 5′ and 3′ junctions. Wild type PGC was used as negative control. (C) Images of donor-PGC derived progeny (I/i) and OVAL targeted progeny after testcross of germline chimera with Korean Ogye (KO) hens. (D) Genomic DNA sequencing analysis of G1 OVAL targeted progenies at 5′ and 3′ junctions amplified by each junction specific primers. PAM sequence is indicated as blue bar and gRNA recognition site is indicated as red bar. Endogenous OVAL gene and donor plasmid sequences are described (E) Pedigree of OVAL targeted chickens. G0 germline chimeric rooster was mated with wild type hen and produced G1 OVAL^±^chickens. G2 OVAL^+/+^, OVAL^±^and OVAL^-/-^ chickens were produced by mating between G1 OVAL^±^individuals. (F) Genotyping of G2 progenies by genomic DNA PCR using specific primers for wild type OVAL allele and modified allele. Primers used for genotyping are indicated as arrows.Fig 1 dummy alt text

To confirm that OVAL^-/-^ chickens do not synthesize OVAL in the oviducts, we performed immunostaining of OVAL in the oviducts of sexually mature OVAL^-/-^ chickens. Histochemical analysis revealed that OVAL was not synthesized in the oviducts of OVAL^-/-^ chickens, but was actively synthesized in the oviducts of OVAL^+/+^chickens (Fig. 2A). The egg white of OVAL^±^chickens showed no visible shape changes (Fig. 2B), and OVAL^-/-^ chicken eggs had a lower egg white volume and egg weight than OVAL^±^ and OVAL^+/+^chicken eggs (Fig. 2B and C). Furthermore, we found that the chick embryos could develop and hatch from OVAL-deficient eggs (OVAL^-/-^), although the fertility and hatchability are significantly lower than those of OVAL^±^ chicken eggs (Fig. 2D). This result suggests that OVAL-deficient egg white can support embryo development, but not as well as OVAL-containing egg white.Fig. 2Characterization of eggs laid by OVAL targeted hens. (A) Immunohistochemistry of Ovalbumin in oviduct of 50 weeks after hatch OVAL^+/+^ and OVAL^-/-^ chickens. Scale bar = 500 μm. (B) Morphology of egg white and egg yolk from eggs of OVAL^+/+^, OVAL^±^and OVAL^-/-^ chickens of 50 weeks after hatch. (C) Measurements of egg weight (n = 6 for each group), egg yolk weight (n = 6 for each group) and egg white volume (n = 6 for each group) of OVAL^+/+^, OVAL^±^and OVAL^-/-^ chicken eggs of 50 weeks after hatch. (D) The hatching rate of progenies from eggs laid by OVAL^±^and OVAL^-/-^ hens. (E) Coomasie-blue staining of OVAL^+/+^, OVAL^±^and OVAL^-/-^ egg white proteins after SDS-PAGE. Major egg white protein bands are indicated as arrows. (F) Western blot analysis of major egg white proteins in egg white of OVAL^+/+^, OVAL^±^and OVAL^-/-^ chickens of 50 weeks after hatch. (G) The concentrations of major egg white proteins in egg white of OVAL^+/+^, OVAL^±^and OVAL^-/-^ chickens of 50 weeks after hatch (n = 3 for each group). Differences among groups were determined by one-way ANOVA. * P < 0.05, ** P < 0.01, and *P< 0.001. ns, non-significant. Bars indicate the SD.Fig 2 dummy alt text

Subsequently, we performed SDS-PAGE and Coomassie blue staining of OVAL^-/-^ and OVAL^+/+^ chicken eggs to quantify major egg white protein composition. The results showed that OVAL was completely absent in egg white of OVAL^-/-^ chickens, but accumulated normally in egg white of OVAL^±^ and OVAL^+/+^ chickens (Fig. 2E). The removal of OVAL was further confirmed by western blot analysis of egg white of OVAL^-/-^ chickens (Fig. 2F). The concentration of OVAL was 76.66 ± 18.36 mg/ml (n = 3) in egg white of OVAL^+/+^ chickens, 33.68 ± 0.44 mg/ml (n = 3) in egg white of OVAL^±^ chickens, and could not be detected in egg white of OVAL^-/-^ chickens (n = 3) (Fig. 2G). This result showed that the quantity of OVAL is reduced when one allele is disrupted, and that OVAL can be completely removed in homozygous knockout chicken eggs. Interestingly, we found that the concentrations of other major egg white proteins tended to increase when the OVAL allele was disrupted. The concentrations of Ovotransferrin were 22.19 ± 4.95 mg/ml (n = 3), 39.12 ± 1.60 mg/ml (n = 3), and 65.64 ± 7.56 mg/ml (n = 3) in egg white of OVAL^+/+^, OVAL^±^, and OVAL^-/-^ chickens, respectively. The concentrations of Lysozyme were 1.28 ± 0.32 mg/ml (n = 3), 1.50 ± 0.09 mg/ml (n = 3), and 2.89 ± 0.67 mg/ml (n = 3) in egg white of OVAL^+/+^, OVAL^±^, and OVAL^-/-^ chickens, respectively. The concentrations of Ovomucin were 2.76 ± 0.12 mg/ml (n = 3), 3.27 ± 0.39 mg/ml (n = 3), and 4.21 ± 0.18 mg/ml (n = 3) in egg white of OVAL^+/+^, OVAL^±^, and OVAL^-/-^ chickens, respectively (Fig. 2G). These results suggest that the compensatory accumulation of proteins in response to the removal of OVAL.

Based on our observation, the most prominent changes resulting from the absence of OVAL are a reduction of egg white volume and increased concentration of other egg white proteins. In egg white morphology, no obvious abnormalities were observed other than the decreased volume, suggesting that the physicochemical properties of the egg white are not substantially altered. With respect to bioactivity, the reduced embryonic development and hatching rates may indicate a decrease in bioactivity of OVAL-deficient egg white. However, this phenotype could also be explained, at least in part, by the reduced volume of egg white rather than by intrinsic functional defects.

In summary, to our knowledge, we report the production of OVAL-null chickens and OVAL-deficient eggs for the first time. This finding challenges the prevailing assumption regarding the indispensable role of OVAL in avian embryogenesis. These results show that genome editing technologies are a useful tool to modulate egg white components for use of eggs as recombinant protein bioreactors or production of hypoallergenic eggs.

Disclosures

The authors declare no conflict of interests.

Declaration

The Authors declare no conflict of interests

CRediT authorship contribution statement

Jin Se Park: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Young Min Kim: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Hong Jo Lee: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Jae Yong Han: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.