Generation of Knock‐In Syrian Hamsters via Zygote Microinjection Using CRISPR/Cas9 Genome Editing

ABSTRACT

Syrian hamsters ( Mesocricetus auratus ) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristics. Although embryo manipulation techniques such as embryo collection, pronuclear microinjection, and embryo transfer have been established, gene knock‐in (KI) hamsters have not yet been reported. Here, we report the successful generation of gene KI Syrian hamsters by microinjecting CRISPR/Cas9 components and plasmid DNA into pronuclear‐stage zygotes. Targeted insertion of a DNA cassette up to 8 kb was achieved at the ROSA26 orthologous locus and other genomic sites. Importantly, we confirmed functional expression of a reporter cassette inserted at the ROSA26 site, providing evidence of transcriptional activity at this locus in Syrian hamsters. Furthermore, we demonstrated that frozen‐thawed KI embryos could give rise to live offspring using a simplified freezing and thawing protocol originally developed for mice and rats. These results confirm the feasibility and applicability of advanced genome editing technologies in Syrian hamsters. These technological advancements enable the development of versatile KI models for applications such as gene expression monitoring and conditional mutagenesis, thereby expanding the utility of Syrian hamsters as model organisms, comparable to mice and rats.

1. Introduction

Syrian hamsters ( Mesocricetus auratus , hereafter termed hamsters), also known as Golden hamsters, are members of the Cricetidae family. They are known for their regular 4‐day sexual cycle, and they produce a substantial amount of vaginal discharge after ovulation, which facilitates the determination of their sexual cycle (Whittaker 1999). Additionally, they have a short gestation period of only 16 days, compared to other rodents such as mice and rats, and can be maintained in the same environment as mice and rats. Because of these characteristics, hamsters have been widely used as an important animal model in many research fields. In particular, hamsters exhibit pharmacological responses that are not shared by other rodents, making them an excellent model for human disease research, including oncology, immunology, and physiology (Juelich et al. 2023; Wang and Cormier 2022; Watanabe et al. 2013). Moreover, hamsters are known as hibernators and serve as useful models in hibernation studies due to their ease of acquisition and breeding, especially when compared to other hibernators (Coussement et al. 2023; Nakagawa and Yamaguchi 2023). Hamsters have also contributed to the understanding of mammalian fertilization mechanisms due to their technical superiority in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Yanagimachi and Chang succeeded in IVF, advancing research on sperm fertilization, acrosome reaction, and sperm–oocyte interaction in mammals (Yanagimachi and Chang 1963). In the 1970s, that group also established the ICSI technique using hamsters (Uehara and Yanagimachi 1976). However, hamster embryos are sensitive to in vitro conditions such as CO₂ concentration, temperature, and artificial light, which can cause early embryonic development to halt during in vitro manipulation (Hirose et al. 2020). Notably, even a 15‐min exposure to artificial light can cause developmental arrest at the 2‐cell stage (Takenaka et al. 2007). To overcome these limitations, some groups have investigated potential mechanisms underlying embryonic arrest (Bavister 1989; Takenaka et al. 2007), embryo culture medium (Barnett and Bavister 1992; Bavister and Arlotto 1990; Schini and Bavister 1988), and procedural conditions (Yamauchi et al. 2002). Through these efforts and the advent of genome editing technologies, it has recently become possible to generate genetically engineered hamsters, and several studies have reported that (Fan et al. 2014; Guo et al. 2020; Hasuwa et al. 2021; Li, Miao, Fan, et al. 2018; Li, Miao, Tabaran, et al. 2018; Miao et al. 2023; Taylor et al. 2022). In addition, the development of the i‐GONAD (improved Genome‐editing via Oviductal Nucleic Acids Delivery) method, which enables genome editing of embryos within the oviduct, has proven effective for hamsters due to their sensitivity to the in vitro environment (Hirose et al. 2020). However, gene editing studies conducted to date have been limited to gene knock‐out (KO), and there have been no reports of gene knock‐in (KI) in hamsters.

In this study, we successfully generated gene cassette KI Syrian hamsters by pronuclear microinjection using CRISPR/Cas9 genome editing. Furthermore, by applying cryopreservation techniques previously established in mice and rats to Syrian hamsters, we demonstrated that cryopreserved and thawed embryos are capable of developing into live offspring. To our knowledge, this is the first report of successful gene cassette KI in hamsters. The development of KI technologies is expected to accelerate in vivo gene function analysis in this species, which has historically trailed behind that of other rodent models such as mice and rats, and to contribute to new advances in medical and biological research.

2. Materials and Methods

2.1. Animals

Golden (Syrian) hamsters were purchased from Japan SLC Inc., housed under specific pathogen‐free (SPF) conditions with controlled lighting conditions (daily light period, 7:00–21:00), at 22°C ± 2°C and 40%–60% humidity (Whittaker 1999). Animals were provided with water and food ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee of RIKEN Kobe Branch.

2.2. Embryo Manipulation Environment

Hamster preimplantation embryos are highly sensitive to artificial light, particularly to short‐wavelength components, which can adversely affect their development (Bavister 1989; Takenaka et al. 2007). To minimize light‐induced damage during embryo collection, microinjection, and transfer, all procedures were performed in a darkroom illuminated with Red LED lights (LED light bulb mini ball Red and Clip light, OHM Electric, Japan), and illuminance was maintained below 8 Lx using a light meter (LX‐2500, CUSTOM, Japan) (Supporting Information Figure S1a–b′). The microscopes used for embryo collection and microinjection in these procedures were equipped with red filters (SUMIPEX TS ST‐115, Sumika Acryl, Japan) (Supporting Information Figure S1c–d′), and for embryo transfer, a red LED ring light (High angle ring lighting, I.P. SYSTEM, Japan) (Supporting Information Figure S1e,e′) was used. Both pieces of equipment block wavelengths shorter than 600 nm. These lighting conditions were implemented to ensure a safe environment for hamster embryos during ex vivo handling. The procedures of embryo manipulation were modified based on previous reports (Fan et al. 2014; Hasuwa et al. 2021; Li, Miao, Fan, et al. 2018; Takenaka et al. 2007; Taylor et al. 2022).

2.3. Zygote Collection

To collect zygotes, female hamsters in proestrus and fertile males were mated at 4 pm. The following morning at 9 am, mating was confirmed by checking for the presence of a vaginal plug or sperm in the vaginal discharge. The vaginal discharge of each hamster was collected using cotton swabs, smeared onto glass slides, and examined using an inverted microscope (CKV41, OLYMPUS, Japan) for the presence of sperm. The oviducts of hamsters that had copulated were excised, and the oviducts were flushed with hamster embryo culture medium (HECM‐9) (Fan et al. 2014; Seshagiri and Vani 2019) containing 0.1% hyaluronidase (prepared in‐house), which had been equilibrated at 37.5°C under 10% CO₂, 5% O₂, and 85% N₂ in an incubator (Multi‐Gas Incubator, Astec, Japan). The pronuclear stage zygotes were collected and cultured in 50 μL drops of HECM‐9 covered with liquid paraffin (Paraffin Liquid, Nacalai Tesque, Japan) until used for microinjection.

2.4. Donor Vectors

In the mouse genome, the ROSA26 locus harbors a non‐coding RNA located between the Setd5 and Thumpd3 genes. As the ortholog of that non‐coding RNA has not been identified in the hamster genome, the genomic organization of Setd5 and Thumpd3 was examined using the golden hamster genome browser (NIH, https://www.ncbi.nlm.nih.gov/gdv/browser/genome/?id=GCF_000349665.1). That gene arrangement was found to be conserved between mice and hamsters. The genomic sequence between Setd5 and Thumpd3 in the hamster genome was compared with the homology arms of the mouse ROSA26 locus previously used for CRISPR‐mediated KI (Abe et al. 2020), and a conserved region was used. Donor vectors targeting the putative hamster ROSA26 locus were constructed by inserting gene cassettes between the homology arms (322 bp and 335 bp), which were cloned into the pBluescript SKII vector. For the R26R‐CAG‐H2B‐Scarlet donor vector, the following components were inserted between the homology arms: a CAG promoter (Niwa et al. 1991), a loxP‐flanked PGK‐Nep‐triple polyA cassette (pBigT) (Srinivas et al. 2001), H2B‐Scarlet (Abe et al. 2011; Bindels et al. 2017) (Addgene plasmid #85044), the woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE), and the bovine growth hormone polyA signal (bpA). For the R26‐H2B‐Scarlet donor vector, the gene cassette included an adenovirus splicing acceptor (SA), H2B‐Scarlet, WPRE, and bpA, inserted between the same homology arms. Donor plasmids were purified using an endotoxin‐free plasmid DNA isolation kit (NucleoBond Xtra Mid EF, TaKaRa, Japan) and stored at −20°C until used. Additional donor vectors (designated CDB0001H, CDB0002H, and CDB0003H) were also generated. The sizes of the homology arms and the inserted DNA cassettes were as follows: CDB0001H, 322 bp/335 bp (same as R26‐H2B‐Scarlet) with an 8 kbp insert; CDB0002H, 356 bp/308 bp with a 1.4 kbp insert; CDB0003H, 319 bp/378 bp with a 1.4 kbp insert. Further details of these constructs and their associated hamster phenotypes will be reported elsewhere.

2.5. RNA Synthesis

gRNA sites were designed using CRISPRdirect (Naito et al. 2015). R26‐crRNA1 (5′‐UCU GGG CCA ACG AUC AAC UAg uuu uag agc uau gcu guu uug‐3′), R26‐crRNA2 (5′‐AUA CCU CCU UAG UUG AUC GUg uuu uag agc uau gcu guu uug‐3′), and tracrRNA (5′‐AAA CAG CAU AGC AAG UUA AAA UAA GGC UAG UCC GUU AUC AAC UUG AAA AAG UGG CAC CGA GUC GGU GCU‐3′) were purchased from FASMAC. crRNAs for other loci, designated with unique project accession numbers (CDB0002H, CDB0003H), will be reported elsewhere. crRNA (500 ng/ul) and tracrRNA (1 μg/μl) were dissolved in RNase‐free water and stored at −80°C until used.

2.6. Microinjection

A microinjection cocktail consisting of Cas9 protein (100 ng/μl) (TrueCut Cas9 Protein v2, Thermo Fisher Scientific, US), R26‐crRNA1 (50 ng/μl), R26‐crRNA2 (50 ng/μl), tracrRNA (200 ng/μL), and a donor vector (10 ng/μl) was used for the CRISPR‐mediated knock‐in. Cre mRNA was injected at a concentration of 1 ng/μl. As in the embryo collection procedure, microinjection was performed in a dark room using a microscope equipped with a red filter (SUMIPEX, Sumika Acryl, Japan) (Supporting Information Figure S1d,d′). Pronuclear stage embryos were placed individually (approximately 10 embryos) in a drop of M2 medium (Sigma‐Aldrich, US). The microinjection cocktail was then injected into the male pronucleus of each embryo using a micromanipulator system (Microscope: DMi8, Leica, Germany; Micromanipulators: TransferMan 4r, Eppendorf, Germany; Microinjector: CellTram 4r, Eppendorf, Germany) and a Piezo driver (PiezoXpert, Eppendorf, Germany) (Abe, Inoue, and Kiyonari 2023). Following microinjection, the embryos were immediately transferred to a drop of HECM‐9 and were maintained in an incubator until transferred.

2.7. Embryo Transfer

Pseudopregnant female hamsters were used as recipients. To induce pseudopregnancy, each vasectomized male was mated with one proestrus female using a cage equipped with a wire net to confirm a plug from the day before microinjection (Abe et al. 2025). Mating was confirmed the following day by the presence of a vaginal plug, either retained within the vagina or found beneath the wire net. Recipient hamsters were anesthetized using a mixture of 0.3 mg/kg medetomidine hydrochloride (Domitor, Nippon Zenyaku Kogyo, Japan), 5 mg/kg butorphanol (Vetorphale, Meiji Seika Pharma, Japan), and 30 mg/kg alfaxalone (Alfaxan, Meiji Seika Pharma, Japan) (MBA). The skin and muscle layers were incised and reflected from the back of the hamster to expose the ovaries and oviducts. The position of the oviduct was confirmed using a stereomicroscope (M80 epi‐illumination stereomicroscope, Leica, Germany) equipped with a red LED ring light (High‐angle ring lighting, I.P. SYSTEM, Japan) (Supporting Information Figure S1e,e′), and approximately 15 injected embryos were transferred into each of the left and right oviducts. After transfer, the hamsters were treated with an antagonist (atipamezole hydrochloride) (Antisedan, Nippon Zenyaku Kogyo, Japan) and were kept warm under a heating lamp (Magnetic Desk Lamp, KANETEC, Japan) until they regained consciousness. Once awake, the hamsters were returned to the breeding room. The recipient hamsters were provided with water agar (Transport agar MEGA, Oriental Yeast, Japan) and Supreme mini‐treats (Supreme mini‐treats, Bio‐Serv, US) daily from the day of embryo transfer until weaning to reduce the risk of pup cannibalism.

2.8. Embryo Images

Embryos were collected at E9.5 and E12.5. Images were captured using a stereomicroscope equipped with epifluorescence optics (M165FC, Leica, Germany) and a camera (DFC310FX, Leica, Germany). A filter set for RFP (excitation: 545/30 nm, emission: 620/60 nm) was used. The images were processed and assembled using software (Photoshop, Adobe Systems, US).

2.9. Genomic DNA Analyses

Tissues from recovered embryos or ears were lysed with proteinase K. After RNA digestion with RNase, DNA was purified by sequential extraction with phenol‐chloroform, followed by ethanol precipitation. The primers used were as follows: R26gtFW2 (P1) (5′‐GGC TTA TCC AAT CCC TAG ACA GAG C‐3′), R26gtREV2 (P2) (5′‐CCA GGT CAT ACT GGA GGA GAT ATC C‐3′), R26gtFW1 (P3) (5′‐AAT GGG AGA CTA GGT GCT CAC CTG G‐3′), CAGgtREV (P4) (5′‐CGC GGA ACT CCA TAT ATGG GCT ATG‐3′), bpA3gtFW (P5) (5′‐GGG GGA GGA TTG GGA AGA CAA TAG C‐3′), R26gtREV1 (P6) (5′‐CAG GCT CAT AGG ACC TTA GGC CAG G‐3′), CAGgtFW (P7) (5′‐CTA CAG CTC CTG GGC AAC GTG CTG G‐3′), PGKgtREV (P8) (5′‐TGT GGA ATG TGT GCG AGG CCA GAG G‐3′), H2BgtREV(P9) (5′‐TTG CGG CTG CGC TTG CGC TTC TTG C‐3′), and SA5gtREV (P10) (5′‐CGG CCT CGA CTC TAC GAT ACC GTC G‐3′). The PCR analyses were performed using primer pairs: the 5′‐side of R26R‐CAG‐H2B‐Scarlet (P3/P4, 534 bp), the 3′‐side of R26R‐CAG‐H2B‐Scarlet (P5/P6, 616 bp), the Neo cassette of R26R‐CAG‐H2B‐Scarlet (P7/P8, 224 bp), the 5′‐side of R26‐CAG‐H2B‐Scarlet (P7/P9, 311 bp), the 5′‐side of R26‐H2B‐Scarlet (P3/P10, 599 bp), the 3′‐side of R26‐H2B‐Scarlet (P5/P6, 537 bp), and the R26 gRNA target site (P1/P2, 338 bp). PCR products were resolved using a microchip electrophoresis system (MultiNa, Shimadzu, Japan). The details of other target loci with unique project accession numbers (CDB0001H, CDB0002H, CDB0003H) will be reported elsewhere.

2.10. Embryo Cryopreservation and Thawing

Embryo cryopreservation and thawing were performed using protocols originally developed for mice or rats. Wild‐type female hamsters in proestrus were paired with wild‐type or R26R‐CAG‐H2B‐Scarlet (KI/+) F1 male hamsters by co‐housing at 16:00. Successful mating was confirmed the following morning. On the morning of the second day post‐coitum, oviducts were excised from females in which mating had been confirmed. Two‐cell stage embryos were collected by flushing the oviducts with HECM9 medium. Embryo cryopreservation was performed according to the methods established for rat (Abe et al. 2025) or mouse (Abe, Inoue, and Kiyonari 2023) embryos. Here, we describe only the rat protocol, which achieved better results for hamster embryos. Collected embryos were placed into a 50 μL drop of P10 medium (ARK Resource, Japan) in a dish, then transferred to the bottom of a cryotube (1.2 mL Inner Serum Tube‐Freestanding, SUMITOMO BAKELITE, Japan) together with 5 μL P10 medium and allowed to stand at room temperature for 10 min. The cryotube was then placed on a cooling plate maintained at −3°C and incubated for 1 min. Subsequently, 95 μL of PEPeS (ARK Resource, Japan), precooled to 0 to −3°C, was added. After standing for 1 min, the tube was plunged into liquid nitrogen. Embryo thawing was performed following a standard mouse embryo thawing protocol (Abe, Inoue, and Kiyonari 2023; Nakao et al. 1997). The cryotube was removed from liquid nitrogen, and residual liquid nitrogen was discarded. The tube was then placed upright at room temperature for 90 s. Following that, 900 μL 0.25 M sucrose in PB1 (prepared in‐house) was added, followed by gentle pipetting. The contents were transferred to a dish, and surviving two‐cell stage embryos were recovered. The recovered embryos were transferred to a 50 μL drop of HECM9 medium covered with liquid paraffin oil and washed through successive drops to remove residual sucrose. Finally, embryos were gathered into a single drop and incubated until transplantation. Thawed two‐cell embryos were transferred into the oviducts of recipient females at 0.5 days post‐coitum.

3. Results

3.1. Generation of Conditional Reporter KI by CRISPR/Cas9‐Mediated Gene Targeting

The mouse ROSA26 locus is widely recognized as a safe harbor locus, which allows the stable and ubiquitous expression of reporter genes without causing any detectable phenotypic abnormalities. The ROSA26 locus has been frequently utilized in gene‐targeting experiments to achieve consistent and reliable reporter gene expression across various tissues and developmental stages in mice and in rats (Abe et al. 2025; Abe et al. 2011). Since the ROSA26 locus had not yet been identified in the hamster genome, we conducted an in silico screening using the hamster genome database. The locus we identified is located between Setd5 and Thumpd3, with a genomic arrangement analogous to the mouse ROSA26 locus. Based on this conserved genomic context and sequence similarity, we concluded that this locus corresponds to the Syrian hamster ROSA26 (shROSA26).

We next attempted to establish a Cre reporter line, R26R‐CAG‐H2B‐Scarlet (6.7 kbp), which allows conditional reporter gene expression upon Cre recombination (Figure 1a). For CRISPR‐mediated KI, a mixture of ctRNP (a ribonucleoprotein complex composed of crRNA, tracrRNA, and Cas9 protein) and the donor vector was microinjected into pronuclear stage zygotes (Figure 1b, Supporting Information Movie S1). The injected zygotes were transferred into the oviducts of pseudopregnant females. From the three rounds of microinjections, 475 out of 482 injected zygotes were transferred into 17 pseudopregnant females, and 7 of those females gave birth to a total of 49 pups (Table 1). Eight of those pups were confirmed to have the KI allele by PCR and sequencing (Figure 1c). Three of them were crossed with wild‐type females, and 5 out of 10, 4 out of 10, and 7 out of 11 of their offspring carried the KI allele, indicating successful germline transmission to the F1 generation. In addition, homozygous KI hamsters generated by crossing F1 KI hamsters were viable and showed no apparent abnormalities.

FIGURE 1.

Generation of R26R‐CAG‐H2B‐Scarlet KI hamsters by CRISPR/Cas9‐mediated microinjection. (a) Strategy for gene cassette KI at the shROSA26 locus. The genomic sequence of the gRNA target site and PAM (protospacer adjacent motif) are shown in blue and red, respectively. The insertion site and PCR primer locations are indicated by an arrowhead and arrows, respectively. HA, homology arm. (b) Snapshot images of a hamster pronuclear‐stage zygote before (upper) and during (lower) microinjection. The red filter was removed for this photo to enhance visibility. Scale bar, 50 μm. (c) Genomic PCR analyses of F0 hamsters. Blue and red arrows indicate the positions of the 5′ and 3′ KI bands, respectively; the black arrow indicates the wild‐type band. Each color corresponds to the arrows in (a). M, DNA ladder marker; WT, wild‐type control. (d) Strategy for conditional Scarlet expression following Cre recombination in R26R‐CAG‐H2B‐Scarlet KI hamsters. Cre‐mediated excision of the loxP‐flanked STOP cassette activates downstream Scarlet expression. PCR primer locations are indicated by arrows. (e) Images of E9.5 Scarlet‐positive and negative embryos after Cre mRNA microinjection. Left panel: Bright field image; right panel: Fluorescence image. Scale bar: 2 mm. (f) Representative genomic PCR results for the STOP and the ΔSTOP alleles. Red and black arrows indicate the positions of the ΔSTOP and STOP bands, respectively. M, DNA ladder marker.

TABLE 1.

Efficiency of CRISPR‐mediated CAG‐H2B‐scarlet KI at the shROSA26 locus.

Insert size	Ex.	No. of injected zygotes	No. of transferred zygotes	Transferred/Injected (%)	No. of pregnant recipients	No. of pups (genotyped)	No. of KI	KI/Pups (%)	KI/Zygotes (%)
6.7 kb	1	153	148	96.7	3/6	15 (14)	1	7.1	0.7
	2	183	182	99.5	1/6	6 (6)	0	0.0	0.0
	3	146	145	99.3	3/5	28 (25)	7	28.0	4.8

Note: Ex.: experimental round; No. of pregnant recipients: number of pregnant females out of the total number of transferred pseudopregnant females; genotyped: number of pups that were successfully genotyped, excluding those that did not survive until genotyping; KI: number of 5′ and 3′ PCR‐positive pups (correct KI); KI/Pups: percentage of knock‐in pups among those that survived until genotyping; KI/Zygotes: percentage of knock‐in pups among injected zygotes.

Furthermore, to confirm the functionality of the conditional allele, we injected Cre mRNA into the cytoplasm of pronuclear stage zygotes obtained from crosses between wild‐type females and F0 monoallelic KI males (Figure 1d). All 39 injected zygotes were transferred into three pseudopregnant females, and embryos were collected at embryonic day 9.5 (E9.5) to observe Scarlet fluorescence. Among the 14 embryos recovered from one female, 5 exhibited ubiquitous Scarlet expression and were confirmed to carry the KI allele by PCR (Figure 1e,f). This result indicates that the loxP‐flanked stop sequence was removed by Cre recombinase, leading to the expression of the Scarlet protein.

3.2. Promoter Activity of the shROSA26 Locus of Hamsters

The mouse ROSA26 was originally identified by a gene trap in embryonic stem cells, in which a β‐geo reporter gene was constitutively expressed during embryonic development (Friedrich and Soriano 1991). However, the promoter activity of this locus has not been confirmed in hamsters. To enhance the future utility of this locus, we investigated its promoter activity by attempting to insert a promoter‐less cassette carrying H2B‐Scarlet (2.2 kbp) into the locus using the same strategy as that employed for R26R‐CAG‐H2B‐Scarlet (Figure 2a). Of the 179 injected zygotes, 177 were transferred into 5 pseudopregnant females, and embryos were collected at E12.5 (Table 2). As a result, one embryo exhibited ubiquitous Scarlet expression throughout its body (Figure 2b) and was confirmed to carry the KI allele by PCR (Figure 2c). These results suggested that the target site exhibits transcriptional activity. Although not addressed in this study, the presence of a non‐coding RNA at this locus remains to be elucidated in future investigations.

FIGURE 2.

Expression of a promoter‐less Scarlet cassette driven by the endogenous shROSA26 promoter. (a) Strategy for promoter‐less gene cassette KI at the shROSA26 locus. PCR primer locations are indicated by arrows. (b) Images of E12.5 Scarlet‐positive and negative embryos. Bright‐field image (left) and RFP fluorescence image (right). Scale bar: 4 mm. (c) Genomic PCR analysis of a wild‐type and a Scarlet‐positive embryo. Blue and red arrows indicate the positions of the 5′ and 3′ KI bands, respectively. M, DNA ladder marker.

TABLE 2.

Efficiency of promoter‐less H2B‐Scarlet KI at the shROSA26 locus.

Insert size	No. of injected zygotes	No. of transferred zygotes	Transferred/Injected (%)	No. of pregnant recipients	No, of E12.5 recovered embryos	RFP (+)	PCR (+)	KI/Embryos (%)	KI/Zygotes (%)
2.2 kb	179	177	98.9	2/5	17	1	1	5.9	0.6

Note: No. of pregnant recipients: number of pregnant females out of the total number of transferred pseudopregnant females; PCR (+): The only RFP‐positive embryo was also 5′ and 3′ PCR‐positive (correct KI); KI/Embryos: percentage of knock‐in embryos among collected embryos; KI/Zygotes: percentage of knock‐in embryos among transferred zygotes.

3.3. Generation of KI Hamsters at Other Loci

To further demonstrate the broader applicability of our method, we attempted to generate additional KI alleles at the shROSA26 locus and two other loci, each with a different size of KI insert (Table 3). Further details of the targeted loci and donor constructs, shown as unique project accession numbers (CDB0001H, CDB0002H, CDB0003H), will be described elsewhere with their phenotypic characterization. The KI efficiency (KI/genotyped pups) of the 8.0 kb insert reporter KI at shROSA26 was 16.7%. In contrast, the KI efficiencies for the smaller 1.4 kb insert were higher, at 53.8% and 53.5% in two independent experiments. This trend of lower efficiency with a larger insert was also observed for 6.7 kb KI (Table 1) in this study. Regardless of these differences in efficiency, our microinjection method successfully generated KI hamsters not only at the shROSA26 locus but also at other loci (Table 3).

TABLE 3.

CRISPR‐mediated KI efficiency at the sh ROSA26 locus and other genomic loci.

Genetic modification	Insert size	No. of injected zygotes	No. of transferred zygotes	Transferred/Injected (%)	No. of pregnant recipients	No. of pups (genotyped)	No. of KI	KI/Pups (%)
CDB0001H	8.0 kb	1042	1030	98.9	5/32	21 (12)	2	16.7
CDB0002H	1.4 kb	438	435	99.3	6/12	30 (13)	7	53.8
CDB0003H	1.4 kb	1018	1010	99.2	14/32	60 (43)	23	53.5

Note: Genetic modification: names of modified genes will be reported elsewhere with their phenotypes; No. of pregnant recipients: number of pregnant females out of the total number of transferred pseudopregnant females; genotyped: number of pups that were successfully genotyped, excluding those that did not survive until genotyping; KI: number of 5′ and 3′ PCR‐positive pups (correct KI); KI/Pups: percentage of knock‐in pups among those that survived until genotyping.

3.4. Cryopreservation of Hamster Embryos

It is crucial that genetically engineered animals be widely shared among research communities worldwide. Cryopreservation technologies for fertilized eggs have greatly facilitated the distribution of mutant mouse and rat strains (Abe et al. 2025; Nakao et al. 1997; Taketsuru and Kaneko 2018). Thus, we sought to explore cryopreservation strategies for hamsters as well. To that end, we compared cryopreservation protocols established for mouse and rat embryos using hamster two‐cell stage embryos (Table 4). For the thawing process, we adopted the well‐established method developed for mouse embryos. A total of 167 fresh (non‐cryopreserved) two‐cell embryos were transferred into six pseudopregnant females, resulting in 20 offspring from five of them (birth rate:13.8% ± 2.7). In the group using frozen embryos prepared with the conventional mouse cryopreservation method, 277 embryos were transferred into nine female hamsters, yielding 26 offspring from seven of them (12.6% ± 3.0). In contrast, 183 embryos cryopreserved using the rat protocol were transferred into seven female hamsters, resulting in 33 offspring from six of them (21.3% ± 3.3). Based on these results, we applied the rat protocol to cryopreserve conditional KI embryos, which subsequently resulted in the successful re‐establishment of KI hamsters (18.6% ± 1.6). In addition, these results demonstrated that hamster embryos cryopreserved by either the mouse protocol or the rat protocol could be successfully reconstituted into live offspring after thawing and transfer.

TABLE 4.

Comparison of embryo cryopreservation methods adapted from mice and rats in Syrian hamsters.

Freezing method	Genotype	No. of frozen 2‐cell	No. of recovered 2‐cell	No. of transferred 2‐cell	No. of pregnant recipients	No. of pups	Birth rate (%)	KI/genotyped
—	Wt	—	—	167	5/6	20	13.8 ± 2.7	—
Mouse	Wt	318	309	277	7/9	26	12.6 ± 3.0	—
Rat	Wt	213	208	183	6/7	33	21.3 ± 3.3	—
Rat	R26R KI	200	196	191	5/7	26	18.6 ± 1.6	13/25

Note: R26R KI: R26R‐CAG‐H2B‐Scarlet; No. of recovered 2‐cell: number of recovered 2‐cell after thawing; No. of transferred 2‐cell: number of recovered 2‐cell that were transferred after thawing, excluding those used in other experiments; No. of pregnant recipients: number of pregnant females out of the total number of transferred pseudopregnant females; Birth rate: percentage of transferred embryos that resulted in live birth, calculated only from recipient females confirmed to be pregnant. Expressed as mean ± standard error.

4. Discussion

In this study, we successfully established a KI genome editing platform for Syrian hamsters using CRISPR/Cas9‐mediated zygote microinjection. Over the past few years, KO hamsters have been generated using either in vivo genome editing via oviductal electroporation (i‐GONAD) (Hirose et al. 2020) or traditional pronuclear microinjection (Fan et al. 2014; Hasuwa et al. 2021; Li, Miao, Fan, et al. 2018; Taylor et al. 2022). However, to our knowledge, no report has demonstrated the successful generation of KI hamsters using either of those approaches. Although i‐GONAD has potential for both KO and KI in hamsters, its feasibility for KI remains unverified, and reproducible protocols have yet to be established. Based on these uncertainties and our accumulated experience with microinjection in multiple species (Abe et al. 2020; Abe et al. 2025; Abe, Kaneko, and Kiyonari 2023; Kiyonari et al. 2021), we selected to use microinjection of pronuclear‐stage zygotes for KI generation in hamsters. While this approach can be limited by the donor DNA size, it enabled successful insertion of large DNA constructs, up to 8 kb. This capacity was critical for establishing conditional reporter lines and for evaluating transcriptional activity from the endogenous shROSA26 locus and allowed us to generate KI at multiple genomic loci. To the best of our knowledge, this is the first report describing the successful generation of large DNA KI hamsters.

First, we aimed to generate conditional reporter KI hamsters by targeting the shROSA26 locus, which is widely used as a safe harbor site in mice and in rats. Using this putative shROSA26 locus, we successfully generated conditional reporter KI hamsters. Upon Cre mRNA microinjection into zygotes derived from these KI hamsters, we observed ubiquitous Scarlet fluorescence in embryos in which the STOP sequence was removed, confirming the Cre‐dependent reporter activation.

Our findings raise the possibility that the shROSA26 locus may possess transcriptional activity, akin to its mouse counterpart. In our study, the observation of widespread fluorescence in E12.5 embryos following insertion of a promoter‐less reporter cassette suggests that this site may be capable of driving gene expression. Although further validation is required, this locus holds promise as a potential safe harbor site for stable transgene integration in hamsters. In addition to the shROSA26 locus, we successfully generated KI alleles at two other genomic loci, demonstrating the broader applicability of our KI strategy in hamsters. Notably, the KI efficiency at those additional loci was higher than that observed at shROSA26, which suggests that the KI efficiency may be affected by factors such as donor DNA size, chromatin accessibility, and locus‐specific genomic context.

In these KI generation experiments, the pregnancy rates following the transfer of microinjected zygotes were relatively low, ranging from 15.6% to 60.0%, with an average of 39.4%. Although the exact cause of this low efficiency remains unclear, it is notable that the pregnancy rate following embryo transfer was markedly lower in the microinjection group compared to the non‐injected control and the freeze‐thawed groups (Table 4, 39.4% vs. 83.3% and 78.3%, respectively). In these experiments, we used a piezo actuator for microinjection, as previously reported for mice and other species (Abe, Inoue, and Kiyonari 2023; Abe et al. 2025; Kiyonari et al. 2021), to minimize damage to the zygotes. However, in the case of hamsters, alternative approaches may need to be considered in the future to improve pregnancy outcomes.

Cryopreservation technique using open‐pulled straw has previously been established in Syrian hamsters (Fan et al. 2016), demonstrating the feasibility of embryo storage and recovery in this species. In the present study, we applied simplified cryopreservation and thawing protocols originally developed for mice and rats and successfully rederived live KI offspring from frozen embryos. This result supports the practicality of adapting rodent protocols for use in hamsters, potentially facilitating broader implementation and distribution of genetically engineered lines.

In this study, all embryos were collected via natural mating without hormonal superovulation. This approach consistently yielded approximately 10 embryos per female. While the use of superovulation in hamsters has been reported in previous studies (Fan et al. 2014; Li, Miao, Fan, et al. 2018), under our conditions, it increased the number of collected embryos but often resulted in a high proportion of unfertilized eggs or inconsistent fertilization outcomes (data not shown). Given that the estrous cycle in hamsters is highly predictable, natural mating provided a more reliable source of fertilized embryos. We therefore opted not to use superovulation. Nonetheless, the development of a reliable superovulation protocol remains important for future work, particularly for improving cryopreservation efficiency, expanding genome editing applications, and ultimately reducing the number of animals required.

The successful generation of KI hamsters in this study marks a significant step forward in the development of genetic engineering tools in this species. While KO models are useful for studying gene loss‐of‐function, KI models uniquely enable precise gene expression control and live imaging using fluorescent reporters. Our results will facilitate the investigation of gene function in vivo, particularly in systems where hamsters exhibit unique biological traits that are not seen in mice. Moreover, the establishment of KI technologies in hamsters opens the door to a wide range of biomedical and basic research applications, such as disease modeling, lineage tracing, and conditional gene regulation.

Author Contributions

M.S., H.K. designed the experiments. M.S., K.I., N.S., A.T., and T.A. performed the experiments and contributed to the preparation of all figures and tables. M.S., T.A., and H.K. wrote the draft of the manuscript. H.K. wrote the final version of the manuscript. All authors read and approved the final manuscript.

Ethics Statement

This article does not contain any studies involving human participants. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of RIKEN Kobe Branch and were conducted in accordance with institutional and national guidelines.

Consent

All authors consent to publication of this article.

Supporting information

Figure S1: Darkroom environment and microscopes used for hamster embryo manipulation. Photographs taken under red light illumination used during embryo manipulation, with room lights turned on (a–e) or turned off (a′–e′). (a, a′) Overview of the overall room lighting setup. (b, b′) Red lighting fixture used to illuminate the working area. (c, c′) Stereomicroscope used for embryo collection. (d, d′) Inverted microscope used for microinjection. (e, e′) Stereomicroscope used for embryo transfer.

Movie S1: Microinjection of hamster pronuclear‐stage zygote. Although a red filter was used during microinjection, this movie was recorded without a red filter to allow for clearer observation.

Shigeta, M. , Inoue K.‐i., Shimada N., Tobe A., Abe T., and Kiyonari H.. 2025. “Generation of Knock‐In Syrian Hamsters via Zygote Microinjection Using CRISPR/Cas9 Genome Editing.” genesis 63, no. 5: e70027. 10.1002/dvg.70027.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.