# CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding

Christophe Richard, Aurélie Allais-Bonnet, Marjolaine André, Valerie Gelin, Marie-Christine Deloche, Dominique Thepot Thépot, Eugénie Canon, Ludivine Laffont, Olivier O. Dubois, Patrice Congar, et al.

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Christophe Richard, Aurélie Allais-Bonnet, Marjolaine André, Valerie Gelin, Marie-Christine Deloche, et al.. CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding. Animal, 2025, 19 (5), pp.101508. 10.1016/j.animal.2025.101508. hal-05058912

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# CRISPR-Cas9 gene-editing to assess the role of RF-amide-related peptide 3 in ovine seasonal breeding

<figure><img src="image_4.png" /></figure>

C. Richard<sup>a,b,1</sup>, A. Allais-Bonnet<sup>a,b,c,1</sup>, M. André<sup>a,b</sup>, V. Gelin<sup>a,b</sup>, M-C. Deloche<sup>a,b,c</sup>, D. Thépot<sup>a,b</sup>, E. Canon<sup>a,b</sup>, L. Laffont<sup>a,b</sup>, O. Dubois<sup>a,b</sup>, P. Congar<sup>d</sup>, O. Lasserre<sup>e</sup>, T. Aguirre-Lavin<sup>e</sup>, G. Gomot<sup>f</sup>, C. Moussu<sup>f</sup>, S. Barbey<sup>f</sup>, E. Pailhoux<sup>a,b,2</sup>, H. Dardente<sup>g,2,*</sup>

## ARTICLE INFO

**Article history:**

Received 5 December 2024

Revised 25 March 2025

Accepted 28 March 2025

Available online 3 April 2025

**Keywords:**

Breeding

Embryo editing

Knock-out

Photoperiodism

Seasonality

## ABSTRACT

Seasonal breeding is an adaptive strategy that ensures the birth of the offspring during the best time of year and allows energy saving in times of food scarcity and adverse environmental conditions. At temperate and polar latitudes, photoperiod is the main synchroniser of seasonal functions, which exerts its action through melatonin. Within the *pars tuberalis* of the anterior pituitary, melatonin triggers a series of events that lead to enhanced local triiodothyronine (T3) production in the medio-basal hypothalamus specifically under long days and ultimately drive the appropriate GnRH output at the median eminence. How T3 governs GnRH output is mostly unknown but neuronal populations that respectively produce KISS1 and RFRP3 appear to be involved. However, while the role of KISS1 as a major GnRH secretagogue is undisputed, the function of RFRP3 in the control of (seasonal) breeding remains enigmatic, with conflicting results hinting at elusive mechanisms. Therefore, we launched an extensive series of experiments in sheep, aimed at invalidating the NPVF gene, which encodes RFRP3, using CRISPR-Cas9 technology. Here, we report on the generation of six sheep for which the NPVF gene has been successfully edited. Four of these animals bear at least one allele expected to behave as a null and constitute founders for distinct ovine lines. These founder sheep will now enter a standard breeding scheme in order to obtain male and female homozygotes for distinct mutations. These animals are expected to provide a clear delineation of the function of RFRP3 in seasonal breeding.

© 2025 The Author(s). Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Implications

The peptide RFRP3 (RF-amide Related Peptide 3), encoded by the NPVF (Neuropeptide VF) gene, might play a key role in the inhibition of breeding during spring-summer in sheep. However, definitive evidence is lacking. To address this question, we used Crispr-Cas9 technology to generate sheep for which the NPVF gene has been edited. These founder animals will enter a standard breeding scheme to obtain male and female homozygotes for

several mutant alleles. According to the working hypothesis, these animals devoid of RFRP3 may breed all year round. This animal model will be useful for basic research purposes only.

## Introduction

Most species exhibit seasonal breeding as an adaptive response to environmental changes. At temperate and polar latitudes, and according to the duration of gestation, seasonal species mate either under short (e.g. sheep) or long days (e.g. hamster species) to allow birth and weaning in late winter/spring, when conditions are most favourable to the survival and growth of parents and progeny. Melatonin secreted during the night by the pineal gland is the endocrine messenger of daylength/photoperiod. A conserved

<footnote><sup>a</sup>Université Paris-Saclay, UVSQ, INRAE, BREED, 78350 Jouy-en-Josas, France</footnote>

<footnote><sup>b</sup> École Nationale Vétérinaire d'Alfort, BREED, 94700 Maisons-Alfort, France</footnote>

<footnote><sup>c</sup>Eliance, Paris, France</footnote>

<footnote><sup>d</sup>INRAE, SAJ, 78350 Jouy-en-Josas, France</footnote>

<footnote><sup>e</sup>INRAE, PAO, Nouzilly, France</footnote>

<footnote><sup>f</sup>INRAE, PIXANIM, Nouzilly, France</footnote>

<footnote><sup>g</sup>INRAE, CNRS, Université de Tours, PRC, 37380 Nouzilly, France</footnote>

<footnote>* Corresponding author.
E-mail address: hugues.dardente@inrae.fr (H. Dardente).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² These authors also contributed equally to this work.</footnote>
molecular cascade linking melatonin action in the *pars tuberalis* of the anterior pituitary to local thyroid hormone signalling in tanycytes of the medio-basal hypothalamus in mammals has been characterised (Hanon et al., 2008; Dardente et al., 2010; Hazlerigg et al., 2024). This mechanism explains the photoperiodic control of local triiodothyronine (T3) production, which governs transitions in most seasonal functions, including breeding (Dardente, 2012). Since T3 increase is invariably tied to long days in all photoperiodic species, the reason for distinct seasonal reproductive strategies between short and long-day breeders must be sought downstream of this common pathway (Helfer et al., 2019; Dardente et al., 2019; Dardente and Simonneaux, 2022), yet upstream of GnRH release at the median eminence.

Here, the neuropeptides RFRP3 (RF-amide Related Peptide 3) and KISS1, produced by distinct clusters of neurons in the DMH/VMH (dorsomedian and ventromedian hypothalamic nuclei) and ARC (hypothalamic arcuate nucleus), have emerged as key players (Dardente and Simonneaux, 2022). A popular model posits that KISS1 and RFRP3 act as central "accelerator and brake", which together pilot GnRH release and seasonal breeding (Kriegsfeld, 2006). There is indeed incontrovertible evidence for the KISS1 stimulatory effect upon GnRH release (Simonneaux, 2020; Pinilla et al., 2012; Beltramo et al., 2014), which drives seasonal breeding in Syrian hamster (Revel et al., 2006) and sheep (Caraty et al., 2007). In contrast, there is still no conclusive evidence that RFRP3 is required for seasonal breeding. RFRP3, produced by the NPVF gene is the mammalian ortholog of GnIH (Gonadotropin Inhibitory Hormone) initially discovered in birds. GnIH/RFRP3 encoding genes have since been identified in all vertebrate groups; birds, amphibians, reptiles, mammals, as well as bony and cartilaginous fishes (Osugi et al., 2014; Tsutsui et al., 2015). GnIH orthologs have also been found in lamprey and amphioxus, which respectively belong to the Agnathans and Protochordates, some of the oldest lineages of the Chordates (Osugi et al., 2014; Tsutsui et al., 2015; Dufour et al., 2020). The NPVF gene and its peptide products are therefore very ancient and have been well-conserved throughout evolution, suggesting an important functional role.

There is evidence that GnIH/RFRP3 inhibits transcription and/or secretion of gonadotropins at the level of the pituitary, but also through a direct action onto GnRH cell bodies, which express the NPFFR1 receptor (cognate receptor for GnIH/RFRP3, Clarke et al., 2008; Smith et al., 2008; Dardente and Simonneaux, 2022). Initial data were consistent with the notion that GnIH/RFRP3 acts as a brake for reproduction. However, further data in fish (especially in some teleosts and cyclostomes) revealed a stimulatory role for GnIH, or failed to find any impact upon gonadotropins release. These findings in fish were soon echoed by data in male Syrian hamster (Ancel et al., 2012) and male mice (Ancel et al., 2017), which showed that RFRP3 activates gonadotropin secretion. Furthermore, the inhibitory impact of RFRP3 in females depends on the stage of the estrus cycle (Kriegsfeld et al., 2006; Henningsen et al., 2017). Interestingly, in male Siberian hamsters, RFRP3 inhibits LH release in long-day exposed, sexually active animals but stimulates LH release in short-day, sexually inactive animals (Ubuka et al., 2012). Furthermore, we performed a thorough set of experiments in sheep, which failed to reveal any impact of RFRP3 upon LH release (Decourt et al., 2016), which contrasts with initial findings claiming a GnIH-like role (i.e. inhibition of LH release; Clarke et al., 2008). Likewise, RFRP3 was found to have no effect upon LH release in pig (Thorson et al., 2015; Lents et al., 2017) and horse (Thorson et al., 2014). Indeed, RFRP3 neurons might not be hypophysiotropic in all mammals (Angelopoulou et al., 2019), which implies that effects seen at the level of the pituitary – usually using pharmacological doses of the peptide – may not be physiologically relevant. Finally,

current data in either NPVF or NPFFR1 KO mice do not provide support for a major role of RFRP3 in reproduction, at least in this non-seasonal breeder (Kiyohara et al., 2017; León et al., 2014). However, expression of NPVF is affected by photoperiod, with higher mRNA levels consistently observed under long days in all photoperiodic species investigated thus far (Angelopoulou et al., 2019; Dardente and Simonneaux, 2022), which suggests that T3 may play a stimulatory role, even though indirectly (Quignon et al., 2020).

Altogether, the role of GnIH/RFRP3 in the control of reproduction remains obscure and the initial simplistic « yin-yang » model based on coordinated opposite actions of KISS1 and RFRP3 needs revision. Furthermore, in addition to photoperiod, NPVF gene expression in sheep is impacted by stress (Clarke et al., 2016), metabolic status (Clarke et al., 2012) and sex steroid feedback (Angelopoulou et al., 2019; Lomet et al., 2020; Dardente and Simonneaux, 2022), which might all be considered as confounding factors when assessing mechanisms of breeding control and might therefore contribute to the discrepancies mentioned above. Finally, ambient temperature (through hypothermia; Jarosławska et al., 2015) also has a profound impact on *Npvf* expression, at least in mice. Considering this and the varied outcomes of experiments in the same species obtained by different teams (especially for sheep) and adding potential issues linked to promiscuity in the receptor signalling of RF-amide peptides (Quillet et al., 2016), we concluded that a genetic strategy was required to unequivocally define the role, if any, of RFRP3 in (seasonal) breeding. We therefore initiated a project in order to obtain – through CRISPR-Cas9 genome editing (GE) – independent sheep lines invalidated for the NPVF gene that encodes RFRP3 (Dardente et al., 2008). The current report details the methodology and outcome of these GE experiments, which led to the generation of six NPVF-edited sheep (three males and three females), four of which are currently being used as founders (F0) for the establishment of independent NPVF-KO lines. Further analysis of these animals will help decipher the role of RFRP3 in seasonal breeding, but also in the adaptation of physiological responses to food challenges, temperature and stress.

## Material and methods

### Animals and facilities

Five experiments, each involving six donor ewes and six recipient ewes, were performed over two consecutive breeding seasons (total of 120 ewes; see Table 1 for calendar). All donor and recipient ewes of Ile-de-France, Romanov and Romane breeds used in this study came from INRAE experimental units (PAO, Nouzilly, France, https://doi.org/10.15454/1.5573896321728955E12 and P3R, Domaine de Galles, Avord, France). All donor ewes (n = 60) and males used for servicing were of the Ile-de-France breed. For Year 1, all recipients' ewes (n = 30) were of the Romanov breed. For Year 2, all recipients' ewes (n = 30) were of the Romane breed. These two breeds were chosen due to their higher prolificity (hence demonstrated ability to carry out gestation of multiple embryos) compared to the Ile-de-France breed, and a better material behaviour, which may ensure a higher survival rate of newborns. Two more INRAE facilities were engaged: the animal experimental units SAJ (Jouy-en-Josas, France) and the Pixanim facility (https://doi.org/10.17180/CQ4D-DW26) attached to the INRAE PRC research unit and dedicated to animal surgery. Donor ewes received hormonal treatments and were exposed to males at the PAO facility before being transported to the SAJ facility using an authorised transporter. Donors ewes were slaughtered at SAJ, ovocytes recovery and gene editing were performed at the BREED research unit. Recipient ewes received hormonal treatments at the PAO facility and were subjected to embryo transfer with
<table><caption>**Table 1**
Number of embryos/oocytes recovered from donor sheep and treated for NPVF genome editing.</caption><thead><tr><th>N° of experiment and date</th><th>Number of embryos / oocytes</th><th>Number of embryos / oocytes put in culture</th><th>Number of two-cells stage embryos</th><th>Cleavage rate (%)</th></tr></thead><tbody><tr><td>#1 – 28 Sep 2021</td><td>58</td><td>57</td><td>25</td><td>43.1%</td></tr><tr><td>#2 – 19 Oct 2021</td><td>47</td><td>46</td><td>7</td><td>15.2%</td></tr><tr><td>#3 – 17 Nov 2021</td><td>55</td><td>54</td><td>37</td><td>68.5%</td></tr><tr><td>#4 – 18 Jan 2022</td><td>35</td><td>30</td><td>22</td><td>73.3%</td></tr><tr><td>#5 – 1 Feb 2022</td><td>91</td><td>86</td><td>32</td><td>37.2%</td></tr><tr><td>#6 – 20 Sep 2022</td><td>40</td><td>38</td><td>18</td><td>47.4%</td></tr><tr><td>#7 – 11 Oct 2022</td><td>44</td><td>44</td><td>9</td><td>20.5%</td></tr><tr><td>#8 – 8 Nov 2022</td><td>90</td><td>87</td><td>59</td><td>67.8%</td></tr><tr><td>#9 – 29 Nov 2022</td><td>65</td><td>61</td><td>35</td><td>57.4%</td></tr><tr><td>#10 – 20 Dec 2022</td><td>40</td><td>37</td><td>26</td><td>70.3%</td></tr><tr><td>Total</td><td>565</td><td>540</td><td>270</td><td>50.0%</td></tr></tbody></table>

fertilised / GE embryos at Pixanim. All founder animals and their offspring were born and raised at PAO.

## Hormonal treatments of donor ewes for production of one-cell embryos

The hormonal treatments of embryo donor ewes followed a "Day 0 Protocol" adapted from Menchaca et al., 2010; Menchaca et al., 2018 and Mittleider et al., 2021 (Fig. 1, upper part). The treatment of donor ewes began with the insertion of an intravaginal sponge (FGA: Fluorogestone Acetate, 30 mg) on day -9. On day -3, the intravaginal sponge was removed and injections of PMSG (Syncro-part®, 600 IU) and PGF2α (Cloprostenol, Prostaglandin F2 alpha or Estrumate®, 1 ml) were administered. Twenty-four hours later (day -2), an injection of GnRH (Gonadotropin-Releasing Hormone, Cystorelin®, 1 ml) was given. Ovulation was predicted to occur 2 days later, marking day 0 and the second phase of the treatment. This began with the insertion of an intravaginal progesterone sponge (FGA: Fluorogestone Acetate, 30 mg). Concurrently, in late afternoon of Day 0, an FSH injection was done (Stimufol®; Reprobiol, Ouffet, Belgium; one vial contains 500 mg of porcine FSH, resuspended with 32 ml of Ringer). This FSH injection was the first in a series of eight. Injections with decreasing doses were then performed twice daily (am and pm) on days 1-3 and in the morning of day 4 (Fig. 1, upper part). On day 3, the FGA sponge

was removed and an injection of PGF2α (0.5 ml) was given in the afternoon. On day 4, ewes further received an injection of PGF2α (0.5 ml) and a final injection of GnRH analogue (Cystorelin®, 1 ml) in the afternoon. On day 5, ewes (group of six) were exposed to a group of three sexually active males in the morning and then to another group of three different sexually active males in the evening. Donor ewes were slaughtered on day 6; their genital tract was immediately removed and dissected, and oviducts were flushed with 20 ml of sterile Euroflush medium (IMV technologies, 019450). The ovulation points were counted on the ovaries and the embryos/oocytes were selected under the stereomicroscope.

## Attempt to select the best donor ewes using serum Anti-Müllerian Hormone levels

For the 1st year, we decided to select donor ewes on the basis of circulating Anti-Müllerian Hormone (AMH) levels, which are expected to be positively correlated with the number of ovocytes that can be ovulated (Monniaux et al., 2011) and their overall quality for embryo production (Pinto et al., 2018). To this end, blood samples were drawn from sixty age-matched ewes of our flock and AMH levels were assessed using a commercial ELISA kit (AnshLabs® Ovine AMH ELISA AL-155; assay sensitivity: 0.025 ng/ml and intra-assay CV < 6.5%). The thirty ewes with the highest AMH levels (in ng/ml, 4.55 ± 1.54 vs 1.59 ± 0.49,

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Schematic representation of the hormonal treatments of donor and recipient ewes. PGF2a: Prostaglandin F2 alpha, Estrumate®; PMSG: Pregnant Mare Serum Gonadotropin, Syncro-part®; GnRH: Gonadotropin-Releasing Hormone, Cystorelin®; hCG: human Chorionic Gonadotropin, Chorulon®.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Number of embryos/oocytes recovered in relation with circulating AMH levels. (A) 60 ewes were sampled and assayed for AMH levels. The 30 ewes with the highest AMH levels were used as donor ewes, the other 30 ewes were returned to the flock. (B) AMH levels measured in donor ewes of experiment #1 to #10 and plotted on x-axis. The number of embryos/oocytes recovered for each donor goat is plotted on the y-axis. No correlation was found. AMH: Anti-Müllerian Hormone. ****P < 0.0001.</figcaption></figure>

respectively for the “high” and “low” AMH groups; range 2.63–8.44 vs 0.50–2.35, respectively) were selected as donors (Fig. 2A). Selecting donor ewes on the basis of the circulating AMH level did not have any impact on the recovery rate of embryos as the coefficient of determination for the thirty ewes of our selected “high” AMH levels group is close to zero (r2 = 0.024; Fig. 2B). Consequently, AMH levels were not assayed to select donor ewes for Year 2 experiments.

### Ewes single-cell embryo treatments using sgRNA/Cas9 ribonucleoprotein complexes

The presumptive zygotes recovered *in vivo* 18 h after last natural insemination, before DNA synthesis to 2-cells occurred, were denuded and injected under the *zona pellucida* with a complex containing (i) a pair of single strand RNA guides (sgRNAs) located within exon 2 (Fig. 3A and 3B) in order to delete most of this exon encoding the peptides RFRP1 and RFRP3 (Year 1) OR (ii) a single sgRNA located immediately downstream of the start-codon of exon 1 on Year 2 (Table 2, Fig. 3A and C) and the TrueCut Cas9 v2 (Thermofischer) by micromanipulation in embryo holding medium (EHM, Ref. 019449, IMV Technologies). All sgRNAs were designed by using the CRISPOR software (https://crispor.gi.ucsc.edu/), purchased at IDT (Integrated DNA Technologies, USA) and used at a concentration of 100 ng/µl each with a ratio sgRNA/Cas9 of 1/0.8. The equipment used for microinjection was Leica DMi8 microscope, femtoJet4i and transferman4r from Eppendorf. After injection, presumptive zygotes were immediately electroporated using a NEPA21 type II Electroporator apparatus (NEPA GENE). This protocol combined both micromanipulation, breaching the *zona pellucida* barrier, and electroporation of recently fertilised zygotes, to deliver GE components, allowing excellent survival of embryos as described in mice (Bagheri et al., 2018). Presumptive zygotes were cultured in 50 µL droplets of a synthetic oviductal fluid medium (Tervit et al., 1972) and incubated at 38.5 °C in a humidified atmosphere containing 5% CO2 and 5% O2. This step was realised on the morning of day 6 (Fig. 1, upper part) and is the starting point of culture. Cleavage was then assessed 18–20 h later, and cleaved embryos were isolated and placed in straws at 35 °C until their transfer into recipient ewes. For Year 1 only, the efficiency of sgRNA was evaluated *in vitro* using embryos derived from bovine ovaries that were recovered in slaughterhouse. This also allowed to reduce the number of donor ewes following the 3R rules. The pair of sgRNAs was designed in DNA regions fully homologous

between ewes and cows (data not shown). This approach assumed that the relative efficiency of GE between the two guides observed in cattle would be similar in ewes. The pair of sgRNAs induced mutations at the NPVF locus in ~90% of injected bovine embryos (n = 60).

### Hormonal treatments of recipient ewes

Day 0 in the treatment timeline of recipient ewes is the same as that for donor ewes (Fig. 1, lower part). On day -8, an intravaginal sponge (Fluorogestone Acetate, 30 mg) was inserted. On the morning of day 2, the recipient ewes received PGF2α (Prostaglandin F2 alpha or Estrumate®, 0.2 ml). Then, on the morning of day 3, the intravaginal sponge was removed and two injections were performed: PMSG (300 IU) and human chorionic gonadotropin (Chorulon®1500, 0.6 ml of a 2 ml solution). Finally, an injection of GnRH (Cystoreline®) was administered on the afternoon of day 4. The recipient ewes were expected to be in estrus on days 6–7. Embryo transfer was performed on the morning of day 7.

### Embryo transfer into recipient ewes

Recipient ewes were fasted for 12 h prior to the surgical procedure. On the day of surgery, anaesthesia was induced by an intravenous injection of Ketamine (Ketamidor®; 10 mg/kg of BW) and Xylazine (Rompun®, 0.01 mg/kg of BW). The animal was placed in a supine position on a pad throughout the procedure. After intubation, anaesthesia was maintained with inhaled isoflurane (1.5–4%), monitored and controlled using a respiratory assistance device. The abdomen was shaved, cleaned with Vetidine® soap and solution, and two incisions were made in the lower abdomen, 3 cm above the udder, in order to introduce laparoscopy tools (camera and forceps to immobilise the ovaries and check for ovulation points). The ovary for which ovulation point was observed was exteriorised using the forceps. The infundibulum of the oviduct was then gently extended using two forceps. Embryos (1–4; mean of ~3.3) were introduced into the oviduct using a sterile micropipette. Once the transfer was complete, incisions were sutured and covered with a protective and healing treatment (Aluspray®). Ewes were then placed in an individual recovery pen under surveillance until awaken. Postoperatively, ewes received an antibiotic treatment (Amoxicillin, Clamoxyl® Suspension; 7 mg/kg of BW for 3 days) and an intramuscular injection of Flunixin for analgesia (Antalzen®, 0.04 ml/kg).
A Gene: NPVF ENSOARG00020000502

neuropeptide VF [Source:NCBI gene (formerly Entrezgene);Acc:100127217]

Gene Synonyms RFRP / Location Primary assembly 4: 72,260,456-72,264,051 forward strand.

ARS-UI_Ramb_v2.0:CM028707.1 / Chr.4

<figure><img src="image_7.png" /></figure>

B

<table><tr><td>1</td><td>ATGGAAATTTCACTAAAACGATTCATTTTATTGATGTAGCCACTTCAAGCTGTTA</td><td>60</td></tr><tr><td>1</td><td>M--E--I--I--S--L--K--R--F--I--L--L--M--L--A--T--S--S--L--L--</td><td>20</td></tr><tr><td>61</td><td>ACATCAAACTCTTCTGTACAGATGAATCAAGGATTTCCAGTCTTTACAGCAAAAGAAT</td><td>120</td></tr><tr><td>21</td><td>F--S--N--I--F--C--T--D--E--S--R--I--P--S--L--Y--S--K--K--N--</td><td>40</td></tr><tr><td>121</td><td>TATGACAAATATCCGAGCctagaggagctagctgggagaagaagaagaagtcttact</td><td>180</td></tr><tr><td>41</td><td>Y--D--K--Y--S--E--P--K--G--D--L--G--W--E--K--K--E--R--S--L--T--</td><td>60</td></tr><tr><td>181</td><td>tttgaagaagtaaaagattgggttccaaaattaagatgaatacacctgccgtcaacaata</td><td>240</td></tr><tr><td>61</td><td>F--E--E--V--K--D--W--G--P--K--I--K--M--N--T--P--A--V--N--K--</td><td>80</td></tr><tr><td>241</td><td>atgccacctctgcaccaacctgccactgattttggagaacatggaagaagaagaagg</td><td>300</td></tr><tr><td>81</td><td>M--P--P--P--S--A--A--N--L--P--L--K--F--G--R--N--M--M--E--E--E--R--</td><td>100</td></tr><tr><td colspan="3"><strong>RFRP1</strong></td></tr><tr><td>301</td><td>agcaactaggtgatggccacctcctctgagaactcgqaaaaataagagaggacagcctt</td><td>360</td></tr><tr><td>101</td><td>S--T--R--V--M--A--H--L--P--L--R--L--G--K--N--R--E--D--S--L--</td><td>120</td></tr><tr><td>361</td><td>tccagacgggtccaaaatctgcccaaaaggtttggaagaacatgagcagccaaagcatc</td><td>420</td></tr><tr><td>121</td><td>S--R--R--V--P--N--L--P--L--K--F--G--R--T--I--A--A--K--S--I--</td><td>140</td></tr><tr><td colspan="3"><strong>RFRP3</strong></td></tr><tr><td>421</td><td>accaagagcctgagttaatttgcctcagcagccatgcatccattccaccatctaccaatgggcta</td><td>480</td></tr><tr><td>141</td><td>F--K--T--L--S--N--L--L--Q--Q--S--M--H--S--P--S--T--N--G--L--</td><td>160</td></tr><tr><td>481</td><td>ctttactccatgacctgcggcccaagaaatccagaatcctggtcaaaagaacctaag</td><td>540</td></tr><tr><td>161</td><td>L--Y--S--M--T--C--R--P--Q--E--I--Q--N--P--G--Q--K--N--L--R--</td><td>180</td></tr><tr><td>541</td><td>AGACTGGGATTCAGCAAAATAGATGATGCAGAATTGAAACAAGAAAATAA</td><td>591</td></tr><tr><td>181</td><td>R--L--G--F--Q--K--I--D--D--A--E--L--K--Q--E--K--*-</td><td>196</td></tr></table>

C

<table><caption>**Fig. 3.** Strategy for editing the sheep NPVF gene. (A) Schematic structure of the ovine NPVF gene and location on chromosome 4. The gene comprises three exons, which all contribute to the CDS. (B) The ORF of the NPVF gene comprises 591 base pairs (exons 1 and 3 are indicated with upper cases, exon 2 with lower cases) and encode a 196 amino acid precursor. The sequence encoding the neuropeptides RFRP1 and RFRP3 is entirely comprised within exon 2. Dashed boxes and arrows indicate location and strand of upper and lower sgRNAs used for Year 1. Sequences of the C-term end of the two RF-amide peptides (LPXRF) are indicated by solid red boxes. (C) Dashed box and arrow indicate the location and strand of the sgRNA used for Year 2. CDS: coding sequence; ORF, open reading frame; sgRNA, single guide RNA.</caption><tr><td>1</td><td>ATGGAAATTATTCACTAAAACGATTCATTTTATTGATGTAGCCACTTCAAGCTGTTA</td><td>60</td></tr><tr><td>1</td><td>M--E--I--I--S--L--K--R--F--I--L--L--M--L--A--T--S--S--L--L--</td><td>20</td></tr><tr><td>61</td><td>ACATCAAACTCTTCTGTACAGATGAATCAAGGATTTCCAGTCTTTACAGCAAAAGAAT</td><td>120</td></tr><tr><td>21</td><td>T--S--N--I--F--C--T--D--E--S--R--I--P--S--L--Y--S--K--K--N--</td><td>40</td></tr><tr><td>121</td><td>TATGACAAATATCCGAGCctagaggagctagctgggagaagaagaagaagtcttact</td><td>180</td></tr><tr><td>41</td><td>Y--D--K--Y--S--E--P--R--G--D--L--G--W--E--K--E--R--S--L--T--</td><td>60</td></tr><tr><td>181</td><td>tttgaagaagtaaaagattgggttccaaaattaagatgaatacacctgccgtcaacaata</td><td>240</td></tr><tr><td>61</td><td>F--E--E--V--K--D--W--G--P--K--I--K--M--N--T--P--A--V--N--K--</td><td>80</td></tr><tr><td>241</td><td>atgccacctctgcaccaacctgccactgattttggagaacatggaagaagaagaagg</td><td>300</td></tr><tr><td>81</td><td>M--P--P--S--A--A--N--L--P--L--K--F--G--R--N--M--E--E--E--R--</td><td>100</td></tr><tr><td>361</td><td>tccagacgggtccaaaatctgcccaaaaggtttggaagaacatgagcagccaaagcatc</td><td>420</td></tr><tr><td>121</td><td>S--R--R--V--P--N--L--P--L--K--F--G--R--T--I--A--A--K--S--I--</td><td>140</td></tr><tr><td colspan="3"><strong>RFRP1</strong></td></tr><tr><td>301</td><td>agcaactaggtgatggccacctcctctgagaactcgqaaaaataagagaggacagcctt</td><td>360</td></tr><tr><td>101</td><td>S--T--R--V--M--A--H--L--P--L--R--L--G--K--N--R--E--D--S--L--</td><td>120</td></tr><tr><td>361</td><td>tccagacgggtccaaaatctgcccaaaaggtttggaagaacatgagcagccaaagcatc</td><td>420</td></tr><tr><td>121</td><td>S--R--R--V--P--N--L--P--L--K--F--G--R--T--I--A--A--K--S--I--</td><td>140</td></tr><tr><td colspan="3"><strong>RFRP3</strong></td></tr><tr><td>421</td><td>accaagacctgagttaatttgcctcagcagtcagccatgcatccattccaccatctaccaatgggcta</td><td>480</td></tr><tr><td>141</td><td>T--K--T--L--S--N--L--L--Q--Q--S--M--H--S--P--S--T--N--G--L--</td><td>160</td></tr><tr><td>481</td><td>ctttactccatgacctgcggcccaagaaatccagaatcctggtcaaaagaacctaag</td><td>540</td></tr><tr><td>161</td><td>L--Y--S--M--T--C--R--P--Q--E--I--Q--N--P--G--Q--K--N--L--R--</td><td>180</td></tr><tr><td>541</td><td>AGACTGGGATTCAGCAAAATAGATGATGCAGAATTGAAACAAGAAAATAA</td><td>591</td></tr><tr><td>181</td><td>R--L--G--F--Q--K--I--D--D--A--E--L--K--Q--E--K--*-</td><td>196</td></tr></table>

**Table 2**

Sheep-specific oligonucleotides used in the study. Bases underlined correspond to the PAM (protospacer adjacent motif). sgRNA: single guide RNA; UTR: untranslated region.

<table><thead><tr><th>Year</th><th>Oligonucleotide</th><th>Location</th><th>5'-3' sequence</th></tr></thead><tbody><tr><td>1</td><td>sgRNA</td><td>exon 2 upstream (+ strand)</td><td>GCCAACCTGCCACTGAGATTTGG</td></tr><tr><td>1</td><td>sgRNA</td><td>exon 2 downstream (- strand)</td><td>CTGCTGGAGCAATTACTCAGGG</td></tr><tr><td>1</td><td>PCR Primer O42O</td><td>upstream - intron 1</td><td>CATTACGTTCCTGCTCCTCG</td></tr><tr><td>1</td><td>PCR Primer O43O</td><td>downstream - intron 2</td><td>GGGCAGGTTCAGTAATTGC</td></tr><tr><td>2</td><td>sgRNA</td><td>exon1 (- strand)</td><td>TGATGTTAACAGCTTGAAAGTGG</td></tr><tr><td>2</td><td>PCR Primer O60P</td><td>upstream - promoter/5' UTR</td><td>ACACGTGGGATTGATTTCCC</td></tr><tr><td>2</td><td>PCR Primer O63P</td><td>downstream - intron 1</td><td>AAACCGTCATGAGACCAGGC</td></tr></tbody></table>

Genotyping of newborns

Newborns were screened for the presence of mutations at the NPVF locus using genomic DNA extracted from ear clips. The targeted regions (exon 2 for Year 1 and exon 1 for Year 2) were amplified by PCR using primers (Table 2) and the Platinum Taq polymerase HiFi (Invitrogen®). The 934 bp and 455 bp PCR amplicons (Year 1 and Year 2, respectively) were cloned and sequenced by Eurofins Genomics, using cloning primers for sequencing (Table 2). Mutations were deduced by comparing the obtained sequence with that of the ovine NPVF genomic locus (see Fig. S1). All mutated alleles were confirmed through cloning (pGEM-T easy, Promega) and sequencing of 6–10 individual clones per GE animal. This refined approach revealed mosaicism as alleles other than the two distinguishable on the initial sequencing chromatogram (be they wild-type or edited) were found for two animals (see Fig. S2).

Results

Design of sgRNAs for gene editing at the ovine NPVF locus

The aim of this study was to generate sheep-bearing null alleles of the NPVF gene, which is located on chromosome 4, comprises 3 exons and encodes a cDNA with a 591 bp open reading frame (ORF) that yields a 196 amino acid precursor protein (neuropeptide VF precursor) from which the neuropeptides RFRP1 and RFRP3 are cleaved (Fig. 3 and Fig. S1 for fully annotated ovine NPVF locus). Experiments were carried out over two consecutive years, using distinct strategies in Year 1 and Year 2 (Table 1, Fig. 3B and Fig. 3C). For Year 1, we aimed at deleting most of exon 2 of the NPVF gene using a pair of sgRNAs that would bring the Cas9 nuclease near the 5' and 3' ends of this exon (Fig. 3B). Since this strategy led to unexpected outcomes (see below), a simplified approach was set for Year 2. A single sgRNA was designed, targeting a
sequence in exon 1 located downstream of the start codon, in order to generate a frame shift in the ORF (Fig. 3C).

## Sheep zygotes recovery and genome editing strategy

All experiments were carried out during the breeding season (autumn and early winter). Overall, in the course of our experiments, sixty embryo donor ewes were used in ten series of experiments over 2 years. Five experiments were carried out between 28 September 2021 and 2 February 2022; the other five were performed during the next reproductive season, from 20 September 2022 to 20 December 2022. In total, 565 one-cell stage embryos or oocytes were recovered, representing an average of 9.4 embryos per ewe (Table 1). Out of the 565 embryos/oocytes manipulated for genome editing, i.e. microinjection followed by electroporation (see Material and Methods), 540 survived this step and were cultured for ~20 h, finally yielding 270 two-cell stage cleaved embryos (Table 1). This represents a cleavage rate of 50%. Embryos/oocytes remaining at the one cell stage are either unfertilised oocytes or embryos blocked consecutively to the in vitro treatments.

## Embryos transfer to recipient ewes and founder animals

Following GE and overnight culture, embryos at the 2-cell stage were selected on gross-morphology criteria, mainly on the appearance of a well-rounded and regular zona pellucida, and on the basis of two blastomers of equivalent size that fully occupy the embryonic volume. Depending on the number of embryos obtained, 1-4 embryos were transferred per recipient ewe. Out of the 270 embryos, 196 were transferred to 60 recipient ewes, with 47 of them (78%) receiving at least three embryos (Table 3). Initial pregnancy diagnosis was performed at 18 days of gestation, G18 (day 0 being the day of servicing), by determining the circulating progesterone levels (Boscos et al., 2003). In 8 out of 10 series, 1-4 recipient ewes were deemed pregnant. Three series (#3, #8 and #9) achieved high success rates with 3-6 ewes having high P4 levels (Table 3). Among the 15 ewes predicted to be pregnant at G18, the pregnancy of 12 of them was confirmed by echography at G45. Each of the twelve ewes naturally delivered one to three newborns at term (Table 3 and Fig. 4). Overall, 17 newborns were obtained, eight males and nine females (Table 3 and Fig. 4). NPVF genotyping by direct DNA sequencing of a PCR amplicon was performed, revealing that six out of these 17 lambs carried at least one GE allele (Table 3 and Fig. 4 and Fig. 5).

For Year 1, no founder animal carrying the expected deletion within exon 2 was obtained (Fig. 4 and Fig. 5A). However, deletions or insertions could be found at both sites (i.e. 5' and 3' ends of exon 2) of sgRNA landing (females #23281 and #23282), which

indicates that Cas9 was properly addressed to these sites but double-strand breaks did not occur concomitantly and non-homologous end joining (NHEJ) occurred rapidly and sequentially. Founder ewes #23281 and #23282 are compound heterozygotes, each bearing two distinct edited alleles of NPVF. For Year 2, we obtained three heterozygous GE animals, two of which (male #33497 and female #33508) are predicted to yield null alleles due to an early stop-codon in the ORF (Fig. 4 and Fig. 5B). The male #33506 bears a large 39 bp deletion, which however preserves the ORF and may therefore not be a null allele. Considering the 2 years, all eight GE alleles carried by the six edited sheep are different, which demonstrates that, beyond their location, mutations induced by Crispr-Cas9 are not predictable (Fig. 4 and Fig. 5). Based on these, crosses between founders and wild-type animals (or backcrosses) are currently ongoing to obtain homozygotes and heterozygotes for the four mutations predicted to yield null alleles (Fig. 4, indicated in red) and assess their impact upon seasonal breeding.

## Discussion

We successfully used CrisPR-Cas9 GE in sheep and obtained six animals, three males and three females, bearing at least one mutant allele at the NPVF locus. Two ewes, #23281 and #23282 are compound heterozygotes for which both alleles are edited. At least four mutant alleles are expected to be null alleles, which should yield animals KO for NPVF with homozygosity. Animals bearing these alleles are currently being used as founders to establish independent sheep lines and clarify the potential role of NPVF and its encoded neuropeptides, RFRP1 and RFRP3, in seasonal breeding.

Over the last ~15 years, various methodologies have been implemented in order to yield efficient GE in mammals: ZFN, Zinc Finger Nucleases; TALEN, Transcription Activator-Like Effector Nuclease. Such strategies are perhaps even more important/relevant to non-model species for which the genetic toolkit is very limited at best. The repurposing of the CrisPR-Cas9 system, and its numerous derivatives, is expected to provide virtually unlimited ability to modify the genome of most species. The scope for use of this methodology in farm animals is particularly broad and has attracted much attention, due to the huge economical impact it might have in decades to come, with a demand for meat products that is expected to rise until at least 2050 (Carlson et al., 2014; Tan et al., 2016; Ruan et al., 2017; Bishop and Van Eenennaam, 2020). There are several areas for which GE has been invoked as a potent tool to improve the genetics of farm animals, which include fighting diseases and providing a sustainable food supply through the introduction of more favourable alleles (Epstein et al., 2021). While chicken, cow and pigs represent the vast majority of meat

<table><caption>**Table 3**
Number of transferred ovine embryos and ewe pregnancies. G stands for Gestation day.</caption><thead><tr><th>N° of experiment and date</th><th>Number of recipient ewes</th><th>Number of transferred embryo</th><th>Predicted pregnant ewes at G18</th><th>Number of pregnant ewes at G45</th><th>Pregnancy rate</th><th>Number of newborns</th><th>Number of founders</th></tr></thead><tbody><tr><td>#1 – 28 Sep 2021</td><td>6</td><td>23</td><td>0</td><td>0</td><td>0%</td><td>0</td><td>0</td></tr><tr><td>#2 – 19 Oct 2021</td><td>6</td><td>7</td><td>1</td><td>1</td><td>16.7%</td><td>1</td><td>1</td></tr><tr><td>#3 – 17 Nov 2021</td><td>6</td><td>24</td><td>3</td><td>3</td><td>50%</td><td>3</td><td>0</td></tr><tr><td>#4 – 18 Jan 2022</td><td>6</td><td>22</td><td>1</td><td>1</td><td>16.7%</td><td>1</td><td>0</td></tr><tr><td>#5 – 1 Feb 2022</td><td>6</td><td>23</td><td>1</td><td>1</td><td>16.7%</td><td>2</td><td>2</td></tr><tr><td>#6 – 20 Sep 2022</td><td>6</td><td>18</td><td>1</td><td>1</td><td>16.7%</td><td>1</td><td>0</td></tr><tr><td>#7 – 11 Oct 2022</td><td>6</td><td>9</td><td>0</td><td>0</td><td>0%</td><td>0</td><td>0</td></tr><tr><td>#8 – 8 Nov 2022</td><td>6</td><td>24</td><td>3</td><td>2</td><td>33.3%</td><td>3</td><td>1</td></tr><tr><td>#9 – 29 Nov 2022</td><td>6</td><td>23</td><td>4</td><td>3</td><td>50%</td><td>6</td><td>2</td></tr><tr><td>#10 – 20 Dec 2022</td><td>6</td><td>23</td><td>1</td><td>0</td><td>0%</td><td>0</td><td>0</td></tr><tr><td>Total</td><td>60</td><td>196</td><td>15</td><td>12</td><td>20%</td><td>17</td><td>6</td></tr></tbody></table>
A?

Upstream sequence - Exon 2

<table><tr><td>WT</td><td>CTCTGCAGCCAACCTGCCACTGAGATTTGGGAGGAACATGGAAG</td></tr><tr><td>20del</td><td>CTCTGCAGCCAACCTGC-------------------ATGGAAG</td></tr><tr><td>17del</td><td>CTCTGCAGCC-------------------TGGGAGGAACATGGAAG</td></tr><tr><td>WT</td><td>CTCTGCAGCCAACCTGCCACTGAG..................ATTTGGGAGGAACATGGAAG</td></tr><tr><td>12ins</td><td>CTCTGCAGCCAACCTGCCACTGAGGAGGAACCTGCCATTTGGGAGGAACATGGAAG</td></tr></table>

Downstream sequence - Exon 2

<table><tr><td>WT</td><td>CATCACCAAGACCCTGAGTAATTTGCTCCAGCAGTCCATGCATT</td></tr><tr><td>8del</td><td>CATCACCAAGACCCT----------TGCTCCAGCAGTCCATGCATT</td></tr><tr><td>3del</td><td>CATCACCAAGACCCTG---AATTTGCTCCAGCAGTCCATGCATT</td></tr><tr><td>WT</td><td>CATCACCAAGACCCTGA.GTAATTTGCTCCAGCAGTCCATGCATT</td></tr><tr><td>1ins</td><td>CATCACCAAGACCCTGAAGTAATTTGCTCCAGCAGTCCATGCATT</td></tr><tr><td>WT</td><td>CATCACCAAGACCCTGAG..TAATTTGCTCCAGCAGTCCATGCATT</td></tr><tr><td>2ins-1sub</td><td>CATCACCAAGACCCTGATTCATAATTTGCTCCAGCAGTCCATGCATT</td></tr></table>

B?

Upstream - Exon 1

<table><caption>**Fig. 4.** Sequences of the ten mutated alleles in Gene-Edited sheep. (A) Alleles obtained in Year 1 (pair of sgRNAs flanking exon 2). Red bold dashes indicate deleted bases (del); red bold letters indicate inserted bases (ins). The wild-type (WT) sequence is shown in light blue. The blue base highlighted in yellow indicates a substituted base. (B) Alleles obtained in Year 2 (a unique sgRNAs within exon 1). Red bold dashes indicate deleted bases (del); bold letters indicate inserted bases (ins). The wild-type (WT) sequence is shown in light blue. Note that mutations 7del and 5ins both lead to the appearance of an early stop codon indicated in underlined bold red letters. sgRNA, single guide RNA.</caption><tr><td>WT</td><td>ATTGATGTTAGCCACTTCAAGCTTGTTAACATCAAACATCTTCTG</td></tr><tr><td>7del</td><td>ATTGATGTTAGCCACTT---------GTTAAACATCAAACATCTTCTG</td></tr><tr><td>39del</td><td>ATT------------------------------------CTG</td></tr><tr><td>WT</td><td>ATTGATGTTAGCCACTT.....CAAGCTTGTTAACATCAAACATCTTCTG</td></tr><tr><td>5ins</td><td>ATTGATGTTAGCCACTTGTTAAACAAGCTTGTTAACATCAAACATCTTCTG</td></tr></table>

products, the use of GE in goats and sheep may also be of interest, for instance to generate Prion-resistant animals (Kalds et al., 2019; Allais-Bonnet et al., 2025). There are however a number of key questions – both ethical and technical – which have to be addressed before action can be taken, with great disparities regarding the status of GE animals (are these to be classified as GMOs ?) at the EU level and worldwide (Schmidt et al., 2020).

Beyond such economical and ethical considerations, GE using CrispR-Cas9 provides basic researchers with a powerful tool to investigate the contribution of a given gene to a phenotype of interest. Seasonal breeding is perhaps one of the most pervasive and spectacular adaptation to a changing annual environment. The ability to turn on and off the reproductive axis in order to breed and deliver the offspring at the right time of year is crucial to the survival of most species on Earth. Yet, the molecular underpinnings cannot be identified in classical rodent models (mouse and rats), which do not typically display photoperiodic control of their reproductive axis and breed all year long. The use of quail,

sheep and hamsters has been pivotal to our current understanding of the integrated mechanisms of photoperiodism, which include a conserved neuroendocrine retrograde pathway that goes from specific melatonin-responsive thyrotrophs of the *pars tuberalis* to tanycytes lining the adjacent third ventricle, then back to the hypothalamus. In this model, T3 produced by tanycytes invariably signals long days and is mandatory to the appropriate downstream GnRH output. In mammals, there is incontrovertible evidence that KISS1 is the main activator of GnRH release, while a role for RFRP3 as a brake has been proposed, but remains largely unsubstantiated (see *supra*). A number of pharmacological compounds (e.g. RF9 and GJ14) have been developed in order to assess the potential implication of RFRP3 and its cognate receptor NPFFR1 in breeding but these studies failed to provide unequivocal answers (Caraty et al., 2012; Liu and Herbison, 2014; Min et al., 2015; Kim et al., 2015; Korthanke et al., 2017), mostly due to promiscuity amongst various RF-amide peptides and their preferred receptor subtype (NPFFR1 and NPFFR2, but also KISS1R).
<table><caption>**Fig. 5.** Number of newborns and founder sheep. Grey shading denotes Gene-Edited (GE) animal. Alleles in red are predicted knockout ones. Year 1 and Year 2 animals are highlighted in yellow and light blue, respectively. M: Male; F: Female; WT: wild-type (i.e. no edited allele); del: deletion; ins: insertion; sub: substitution; up: upstream (i.e. 5' site); down: downstream (i.e. 3' site).</caption><thead><tr><th>Recipient ID</th><th>Lamb ID</th><th>Sex</th><th>Number of detected alleles</th><th>Description of alleles</th></tr></thead><tbody><tr><td>16923</td><td>23194</td><td>M</td><td>2</td><td>8 del*down / WT</td></tr><tr><td>15483</td><td>23243</td><td>F</td><td>1</td><td>WT</td></tr><tr><td>16764</td><td>23236</td><td>M</td><td>1</td><td>WT</td></tr><tr><td>58015</td><td>23244</td><td>M</td><td>1</td><td>WT</td></tr><tr><td>16373</td><td>23280</td><td>F</td><td>1</td><td>WT</td></tr><tr><td rowspan="2">58027</td><td>23281</td><td>F</td><td>2</td><td>20 del*up + 1 ins*down / 12 ins*up + 2 ins-1sub*down</td></tr><tr><td>23282</td><td>F</td><td>2</td><td>17 del*up + 3 del*down / 3del*down</td></tr><tr><td>67807</td><td>33009</td><td>F</td><td>1</td><td>WT</td></tr><tr><td rowspan="2">03219</td><td>33498</td><td>M</td><td>1</td><td>WT</td></tr><tr><td>33499</td><td>F</td><td>1</td><td>WT</td></tr><tr><td>94560</td><td>33497</td><td>M</td><td>2</td><td>7 del / WT</td></tr><tr><td>14655</td><td>33501</td><td>F</td><td>1</td><td>WT</td></tr><tr><td rowspan="2">85968</td><td>33507</td><td>M</td><td>1</td><td>WT</td></tr><tr><td>33508</td><td>F</td><td>2</td><td>5 ins / WT</td></tr><tr><td rowspan="3">88838</td><td>33504</td><td>F</td><td>1</td><td>WT</td></tr><tr><td>33505</td><td>M</td><td>1</td><td>WT</td></tr><tr><td>33506</td><td>M</td><td>2</td><td>39 del / WT</td></tr></tbody></table>

Therefore, we decided to use GE to knockout portions of the NPVF gene in sheep in order to prevent the production of a functional mRNA/precursor protein and therefore assess the potential role of RFRP3 in seasonal breeding. Our extensive experimental effort included a total of 10 sessions, each including six donor and six recipient ewes, which spanned over 2 consecutive years, with two different strategies for GE. The overall pregnancy rate was 20% and 17 lambs were born from 12 ewes, including three ewes that delivered two lambs and a single ewe that delivered three lambs. Out of these 17 lambs, 6 presented with GE of NPVF (~35%). Two females (litter mates from the same recipient ewe) were found to be compound heterozygotes, bearing two distinct edited alleles of the NPVF gene. Our initial GE strategy (Year 1) aimed at deleting most of exon 2, which encodes both RFRP1 (whose function, if any, remains unclear) and RFRP3, using a pair of sgRNAs. This strategy did not yield the expected outcome. GE occurred either at the location of a single sgRNA (downstream sgRNA for male #23194) or indeed occurred at both upstream and downstream sites (females #21181 and #23182), as expected, yet did not lead to removal of the fragment of exon 2. The most parsimonious explanation for this might be that Cas9 acted sequentially, cutting first at one end, followed by repair through NHEJ, and then cutting a second time at the other end, before being repaired. For female #23281, this appeared to have occurred concomitantly for both alleles at the zygotic stage, before the first cell division, since the analysis of independent clones did not reveal any other alleles than the two reported in Fig. 5.

The situation appears more complex for female #23282, which further displays mosaicism as revealed by analysis of independent clones (at least four alleles corresponding to three distinct mutations events, including 1sub-3del* down, Fig. 4 and Fig. S2). Male #23194 also displayed mosaicism as an allele harbouring the 8*del down along with a large 60*ins up (preserving the ORF, see Fig. S2) was also found. It is plausible that mosaicism is indeed present in most, if not all, of our GE-edited sheep. However, the exact

number and relative proportion of different alleles present in each animal cannot be determined using the methodologies described here. Deep sequencing of multiple RNA samples from different tissues would be required to provide a clearer account of this, which would be of limited value for our breeding purposes. Indeed, mosaicism is a classical issue when using Crispr-Cas9, as recently observed for the invalidation of KISS1 in pigs for instance (Flórez et al., 2023, and references therein). To avoid complex genotypes and limit mosaicism, we used a simplified approach for Year 2. A single sgRNA was designed within exon 1, ~50 bp downstream of the start codon, in order to obtain insertions or deletions leading to a frame shift and, potentially, appearance of an early stop codon. We obtained three heterozygous animals, two of which (male #33497 and female #33508) possess an edited NPVF allele that yields an early stop-codon.

We initially decided to select donor ewes on the basis of circulating AMH levels, which are expected to be positively correlated with the number of ovocytes that can be ovulated (Monniaux et al., 2011) and their overall quality for embryo production (Pinto et al., 2018). However, in our hands, and in the context of this experiment, circulating AMH levels are not a valuable marker for predicting ovulation rate in ewes. These data are consistent with recent findings in ewes (Brochado et al., 2024) and our own findings in goats (Allais-Bonnet et al., 2025), which failed to find any overt positive correlation between AMH levels and ovulation efficiency in super-ovulation protocols.

As it stands, there are limitations to our study, which need to be carefully considered. While the off-target impact of sgRNA cannot be excluded, the use of four distinct sheep lines, harbouring four different mutations obtained through two different strategies will make it very unlikely that the reproductive phenotype (if any) would be due to mutation at any site other than the NPVF locus. Furthermore, any off-target mutation, should it exist, would only influence the phenotype if physically closely linked to the NPVF locus as it would limit its segregation during reproduction. While
this cannot be excluded, such an event is deemed very unlikely considering the selection criteria applied when generating sgRNA. Furthermore, we wish to mention that the search for potential off-targets, while legitimate, is necessarily limited by the level of completeness of the genome used for your bio-informatics analysis. As it stands, the ovine genome draft is still far from complete. Therefore, any evaluation of off-targets well necessarily be non-exhaustive. Hence, even though cumbersome and lengthy, the generation and phenotypic comparison of independent lines (i.e. bearing distinct mutations at the NPVF locus) appears as a more powerful approach. Another question, which cannot currently be solved, pertains to the impact of the different mutations reported here upon the production of an mRNA and protein/peptides. We deem it very likely that some of these alleles are indeed null alleles (see *supra*), effectively preventing the production of RFRP3. We acknowledge that this will have to be verified using RT-qPCR for the mRNA and ICC or dot-blot for the peptide in tissues known to express the NPVF gene, which means hypothalamus and gonads. However, there is no need to explain that putting down our F0 founders to check this is not an option.

Taken together, our experimental efforts have led to the generation of six GE sheep for the NPVF gene. Four of these animals (females #23281 & #23282 from Year 1, female #33508 & male #33497 from Year 2) will serve as founders to establish independent lines. The research reported here constitutes the first step of an unparalleled effort to tackle an unanswered question of basic physiology using a non-model farm species. The thorough analysis of independent ovine GE lines invalidated for NPVF will allow us to delineate the role of RFRP3 in the control of seasonal breeding.

## Supplementary material

Supplementary Material for this article (https://doi.org/10.1016/j.animal.2025.101508) can be found at the foot of the online page, in the Appendix section.

## Ethics approval

All experimental procedures were performed in accordance with international (directive 2010/63/UE) and national legislation (décret n° 2013-118) governing the ethical use of animals in research (authorisation n° E37-175-2 and n°A38 801). All experiments were performed with the approval of the French Ministry for Higher Education, Research and Innovation, MESRI (accreditation numbers APAFIS#31291-2021042116427698 v3 & APAFIS#31129-2021042116476756 v3) and following the guidelines issued by the Val-de-Loire ethics committee (CEEA VdL N°019). This project using GE in ewes has also received a favourable opinion from the INRAE committee for New Breeding Techniques (NBTs). All scientists working directly with the animals possess an animal experimentation licence delivered by the French veterinary services.

## Data and model availability statement

None of the data were deposited in an official repository. The data/models that support the study findings are available to reviewers, or available from the authors upon request.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the author(s) did not use any AI and AI-assisted technologies.

## Author ORCIDs

Christophe Richard: https://orcid.org/0000-0003-1488-4815.

Aurélie Allais-Bonnet: https://orcid.org/0000-0002-5484-0270.

Marjolaine André: https://orcid.org/0009-0003-1745-1932.

Valérie Gélin: https://orcid.org/0000-0002-4146-4405.

Marie-Christine Deloche: https://orcid.org/0009-0001-9181-8929.

Dominique Thépot: https://orcid.org/0009-0001-1383-1597.

Olivier Dubois: https://orcid.org/0009-0004-4430-3860.

Patrice Congar: https://orcid.org/0000-0002-5691-6582.

Eric Pailhoux: https://orcid.org/0000-0001-8012-9143.

Hugues Dardente: https://orcid.org/0000-0001-7209-5940.

## CRediT authorship contribution statement

C. Richard: Writing - review & editing, Methodology, Investigation. A. Allais-Bonnet: Writing - review & editing, Methodology, Investigation. M. André: Writing - review & editing, Methodology, Investigation. V. Gelin: Writing - review & editing, Methodology, Investigation. M-C. Deloche: Writing - review & editing, Methodology, Investigation. D. Thépot: Writing - review & editing, Methodology. E. Canon: Writing - review & editing, Methodology, Investigation. L. Laffont: Writing - review & editing, Investigation. O. Dubois: Writing - review & editing, Investigation. P. Congar: Writing - review & editing, Project administration. O. Lasserre: Writing - review & editing, Project administration. T. Aguirre-Lavin: Writing - review & editing, Project administration. G. Gomot: Project administration. C. Moussu: Project administration. S. Barbey: Writing - review & editing, Project administration. E. Pailhoux: Writing - review & editing, Writing - original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Conceptualisation. H. Dardente: Writing - review & editing, Writing - original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualisation.

## Declaration of interest

None.

## Acknowledgements

The authors would like to thank Aurélien Raynaud, Alexis Boissonnade and Jean-François Alkombre, and all the staff of the facility (SAJ, INRAE, Jouy-en-Josas, France) for managing the donor ewes. The authors also thank the MIMA2 Imaging Core Facility (Microscopie et Imagerie des Microorganismes, Animaux et Aliments, INRAE, Jouy-en-Josas, https://doi.org/10.15454/1.5572348210007727E12).

## Financial support statement

This research has been funded by the ANR GMOPhen (ANR-20-CE14-0031).

## References

Allais-Bonnet, A., Richard, C., André, M., Gelin, V., Deloche, M.-C., Lamadon, A., Morin, G., Mandon-Pépin, B., Canon, E., Thépot, D., Laubier, J., Moazami-Goudarzi, K., Laffont, L., Dubois, O., Fassier, T., Congar, P., Lasserre, O., Aguirre-Lavin, T., Vilotte, J.-L., Pailhoux, E., 2025. CRISPR/Cas9-editing of PRNP in Alpine goats. Veterinary Research 56, 11. https://doi.org/10.1186/s13567-024-01444-1.
Ancel, C., Bentsen, A.H., Sébert, M.-E., Tena-Sempere, M., Mikkelsen, J.D., Simonneaux, V., 2012. Stimulatory effect of RFRP-3 on the gonadotrophic axis in the male Syrian hamster: the exception proves the rule. Endocrinology 153, 1352-1363. https://doi.org/10.1210/en.2011-1622.

Ancel, C., Inglis, M.A., Anderson, G.M., 2017. Central RFRP-3 stimulates LH secretion in male mice and has cycle-dependent inhibitory effects in females. Endocrinology 158, 2873-2883. https://doi.org/10.1210/en.2016-1902.

Angelopoulou, E., Quignon, C., Kriegsfeld, L.J., Simonneaux, V., 2019. Functional implications of RFRP-3 in the central control of daily and seasonal rhythms in reproduction. Frontiers in Endocrinology 10, 183. https://doi.org/10.3389/fendo.2019.00183.

Bagheri, H., Friedman, H., Shao, H., Chong, Y., Lo, C.-A., Emran, F., Kays, I., Yang, X.-J., Cooper, E., Chen, B.E., Siminovitch, K., Peterson, A., 2018. TIE: a method to electroporate long DNA templates into preimplantation embryos for CRISPR-Cas9 gene editing. The CRISPR Journal 1, 223-229. https://doi.org/10.1089/crispr.2017.0020.

Beltramo, M., Dardente, H., Cayla, X., Caraty, A., 2014. Cellular mechanisms and integrative timing of neuroendocrine control of GnRH secretion by kisspeptin. Molecular and Cellular Endocrinology 382, 387-399. https://doi.org/10.1016/j.mce.2013.10.015.

Bishop, T.F., Van Eenennaam, A.L., 2020. Genome editing approaches to augment livestock breeding programs. The Journal of Experimental Biology 223, webjeb201759. https://doi.org/10.1242/jeb.201759.

Boscas, C.M., Samartzi, F.C., Lymberopoulos, A.G., Stefanakis, A., Belibasaki, S., 2003. Assessment of progesterone concentration using enzymeimmunoassay, for early pregnancy diagnosis in sheep and goats. Reproduction in Domestic Animals = Zuchthygiene 38, 170-174. https://doi.org/10.1046/j.1439-0531.2003.00407.x.

Brochado, C., Duran, B.J., Kieffer, J.D., Pinczak, A., Menchaca, A., Garcia-Guerra, A., 2024. Reduced embryo yield obtained from superstimulated ewes with low circulating AMH concentration is improved by lengthening the FSH treatment. Theriogenology 218, 79-88. https://doi.org/10.1016/j.theriogenology.2024.01.024.

Caraty, A., Smith, J.T., Lomet, D., Ben Saïd, S., Morrissey, A., Cognie, J., Doughton, B., Baril, G., Briant, C., Clarke, I.J., 2007. Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes. Endocrinology 148, 5258-5267. https://doi.org/10.1210/en.2007-0554.

Caraty, A., Blomenröhr, M., Vogel, G.M.T., Lomet, D., Briant, C., Beltramo, M., 2012. RF9 powerfully stimulates gonadotropin secretion in the ewe: evidence for a seasonal threshold of sensitivity. Journal of Neuroendocrinology 24, 725-736. https://doi.org/10.1111/j.1365-2826.2012.02283.x.

Carlson, D.F., Tan, W., Hackett, P.B., Fahrenkrug, S.C., 2014. Editing livestock genomes with site-specific nucleases. Reproduction, Fertility, and Development 26, 74-82. https://doi.org/10.1071/RD13260.

Clarke, I.J., Sari, I.P., Qi, Y., Smith, J.T., Parkington, H.C., Ubuka, T., Iqbal, J., Li, Q., Tilbrook, A., Morgan, K., Pawson, A.J., Tsutsui, K., Millar, R.P., Bentley, G.E., 2008. Potent action of RFamide-related peptide-3 on pituitary gonadotropes indicative of a hypophysiotropic role in the negative regulation of gonadotropin secretion. Endocrinology 149, 5811-5821. https://doi.org/10.1210/en.2008-0575.

Clarke, I.J., Smith, J.T., Henry, B.A., Oldfield, B.J., Stefanidis, A., Millar, R.P., Sari, I.P., Chng, K., Fabre-Nys, C., Caraty, A., Ang, B.T., Chan, L., Fraley, G.S., 2012. Gonadotropin-inhibitory hormone is a hypothalamic peptide that provides a molecular switch between reproduction and feeding. Neuroendocrinology 95, 305-316. https://doi.org/10.1159/000332822.

Clarke, I.J., Bartolini, D., Conducter, G., Henry, B.A., 2016. Stress increases gonadotropin inhibitory hormone cell activity and input to GnRH cells in ewes. Endocrinology 157, 4339-4350. https://doi.org/10.1210/en.2016-1513.

Dardente, H., 2012. Melatonin-dependent timing of seasonal reproduction by the pars tuberalis: pivotal roles for long daylengths and thyroid hormones. Journal of Neuroendocrinology 24, 249-266. https://doi.org/10.1111/j.1365-2826.2011.02250.x.

Dardente, H., Simonneaux, V., 2022. GnRH and the photoperiodic control of seasonal reproduction: delegating the task to kisspeptin and RFRP-3. Journal of Neuroendocrinology 34, e13124. https://doi.org/10.1111/jne.13124.

Dardente, H., Birnie, M., Lincoln, G.A., Hazlerigg, D.G., 2008. RFamide-related peptide and its cognate receptor in the sheep: cDNA cloning, mRNA distribution in the hypothalamus and the effect of photoperiod. Journal of Neuroendocrinology 20, 1252-1259. https://doi.org/10.1111/j.1365-2826.2008.01784.x.

Dardente, H., Wyse, C.A., Birnie, M.J., Dupré, S.M., Loudon, A.S.I., Lincoln, G.A., Hazlerigg, D.G., 2010. A molecular switch for photoperiod responsiveness in mammals. Current Biology: CB 20, 2193-2198. https://doi.org/10.1016/j.cub.2010.10.048.

Dardente, H., Wood, S., Ebling, F., Sáenz de Miera, C., 2019. An integrative view of mammalian seasonal neuroendocrinology. Journal of Neuroendocrinology 31, e12729. https://doi.org/10.1111/jne.12729.

Decourt, C., Anger, K., Robert, V., Lomet, D., Bartzen-Sprauer, J., Caraty, A., Dufourny, L., Anderson, G., Beltramo, M., 2016. No evidence that RFamide-related peptide 3 directly modulates LH secretion in the ewe. Endocrinology 157, 1566-1575. https://doi.org/10.1210/en.2015-1854.

Dufour, S., Quérat, B., Tostivint, H., Pasqualini, C., Vaudry, H., Rousseau, K., 2020. Origin and evolution of the neuroendocrine control of reproduction in vertebrates, with special focus on genome and gene duplications. Physiological Reviews 100, 869-943. https://doi.org/10.1152/physrev.00009.2019.

Epstein, L.R., Lee, S.S., Miller, M.F., Lombardi, H.A., 2021. CRISPR animals, and FDA oversight: building a path to success. Proceedings of the National Academy of Sciences of the United States of America 118, e2004831117. https://doi.org/10.1073/pnas.2004831117.

Flórez, J.M., Martins, K., Solin, S., Bostrom, J.R., Rodríguez-Villamil, P., Ongaratto, F., Larson, S.A., Ganbaatar, U., Coutts, A.W., Kern, D., Murphy, T.W., Kim, E.-S., Carlson, D.F., Huisman, A., Sonstegard, T.S., Lents, C.A., 2023. CRISPR/Cas9-editing of KISS1 to generate pigs with hypogonadotropic hypogonadism as a castration free trait. Frontiers in Genetics 13, 1078991. https://doi.org/10.3389/fgene.2022.1078991.

Hanon, E.A., Lincoln, G.A., Fustin, J.-M., Dardente, H., Masson-Pévet, M., Morgan, P.J., Hazlerigg, D.G., 2008. Ancestral TSH mechanism signals summer in a photoperiodic mammal. Current Biology: CB 18, 1147-1152. https://doi.org/10.1016/j.cub.2008.06.076.

Hazlerigg, D.G., Simonneaux, V., Dardente, H., 2024. Melatonin and seasonal synchrony in mammals. Journal of Pineal Research 76, e12996. https://doi.org/10.1111/jpi.12996.

Helfer, G., Barrett, P., Morgan, P.J., 2019. A unifying hypothesis for control of body weight and reproduction in seasonally breeding mammals. Journal of Neuroendocrinology 31, e12680. https://doi.org/10.1111/jne.12680.

Henningsen, J.B., Ancel, C., Mikkelsen, J.D., Gauer, F., Simonneaux, V., 2017. Roles of RFRP-3 in the daily and seasonal regulation of reproductive activity in female syrian hamsters. Endocrinology 158, 652-663. https://doi.org/10.1210/en.2016-1689.

Jarosławska, J., Chabowska-Kita, A., Kaczmarek, M.M., Kozak, L.P., 2015. Npvf: hypothalamic biomarker of ambient temperature independent of nutritional status. PLoS Genetics 11, e1005287. https://doi.org/10.1371/journal.pgen.1005287.

Kalds, P., Zhou, S., Cai, B., Liu, J., Wang, Y., Petersen, B., Sonstegard, T., Wang, X., Chen, Y., 2019. Sheep and goat genome engineering: from random transgenesis to the CRISPR era. Frontiers in Genetics 10, 750. https://doi.org/10.3389/fgene.2019.00075.

Kim, J.S., Brownjohn, P.W., Dyer, B.S., Beltramo, M., Walker, C.S., Hay, D.L., Painter, G. F., Tyndall, J.D.A., Anderson, G.M., 2015. Anxiogenic and stressor effects of the hypothalamic neuropeptide RFRP-3 are overcome by the NPFFR antagonist GJ14. Endocrinology 156, 4152-4162. https://doi.org/10.1210/en.2015-1532.

Kiyohara, M., Son, Y.L., Tsutsui, K., 2017. Involvement of gonadotropin-inhibitory hormone in pubertal disorders induced by thyroid status. Scientific Reports 7, 1042. https://doi.org/10.1038/s41598-017-01183-8.

Korthanke, C., Thorson, J., Prezotto, L., Welsh, T., Cardoso, R., Williams, G., 2017. Secretion of gonadotropins in response to a novel kiss-1 receptor agonist, RF9 in the mare: modulation by estradiol-17β and half-life of RF9 in the peripheral circulation. Journal of Equine Veterinary Science 57, 100-106. https://doi.org/10.1016/j.jevs.2017.07.013.

Kriegsfeld, L.J., 2006. Driving reproduction: RFamide peptides behind the wheel. Hormones and Behavior 50, 655-666. https://doi.org/10.1016/j.yhbeh.2006.06.004.

Kriegsfeld, L.J., Mei, D.F., Bentley, G.E., Ubuka, T., Mason, A.O., Inoue, K., Ukena, K., Tsutsui, K., Silver, R., 2006. Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals. Proceedings of the National Academy of Sciences of the United States of America 103, 2410-2415. https://doi.org/10.1073/pnas.0511003103.

Lents, C.A., Thorson, J.F., Desaulniers, A.T., White, B.R., 2017. RFamide-related peptide 3 and gonadotropin-releasing hormone-II are autocrine-paracrine regulators of testicular function in the boar. Molecular Reproduction and Development 84, 994-1003. https://doi.org/10.1002/mrd.22830.

León, S., García-Galiano, D., Ruiz-Pino, F., Barroso, A., Manfredi-Lozano, M., Romero-Ruiz, A., Roa, J., Vázquez, M.J., Gaytan, F., Blomenrohr, M., van Duin, M., Pinilla, L., Tena-Sempere, M., 2014. Physiological roles of gonadotropin-inhibitory hormone signaling in the control of mammalian reproductive axis: studies in the NPFF1 receptor null mouse. Endocrinology 155, 2953-2965. https://doi.org/10.1210/en.2014-1030.

Liu, X., Herbison, A.E., 2014. RF9 excitation of GnRH neurons is dependent upon Kiss1r in the adult male and female mouse. Endocrinology 155, 4915-4924. https://doi.org/10.1210/en.2014-1517.

Lomet, D., Druart, X., Hazlerigg, D., Beltramo, M., Dardente, H., 2020. Circuit-level analysis identifies target genes of sex steroids in ewe seasonal breeding. Molecular and Cellular Endocrinology 512, 110825. https://doi.org/10.1016/j.mce.2020.110825.

Menchaca, A., Vilariño, M., Crispo, M., de Castro, T., Rubianes, E., 2010. New approaches to superovulation and embryo transfer in small ruminants. Reproduction, Fertility, and Development 22, 113-118. https://doi.org/10.1071/RD09222.

Menchaca, A., Dos Santos-Neto, P.C., Cuadro, F., Souza-Neves, M., Crispo, M., 2018. From reproductive technologies to genome editing in small ruminants: an embryo's journey. Animal Reproduction 15, 984-995. https://doi.org/10.21451/1984-3143-AR2018-0022.

Min, L., Leon, S., Li, H., Pinilla, L., Carroll, R.S., Tena-Sempere, M., Kaiser, U.B., 2015. RF9 acts as a KISS1R agonist in vivo and in vitro. Endocrinology 156, 4639-4648. https://doi.org/10.1210/en.2015-1635.

Mittleleider, T., Collins, S., Gibbons, P., Gibbons, J., 2021. 12 Artificial insemination and embryo transfer results in ewes during a long daylength period. Reproduction, Fertility and Development 33, 113-114. https://doi.org/10.1071/RDv33n2Ab12.

Monniaux, D., Baril, G., Laine, A.-L., Jarrier, P., Poulin, N., Cognié, J., Fabre, S., 2011. Anti-Mullerian hormone as a predictive endocrine marker for embryo
production in the goat. Reproduction (cambridge, England) 142, 845-854.
https://doi.org/10.1530/REP-11-0211.

Osugi, T., Ubuka, T., Tsutsui, K., 2014. Review: evolution of GnIH and related peptides structure and function in the chordates. Frontiers in Neuroscience 8, 255. https://doi.org/10.3389/fnins.2014.00255.

Pinilla, L., Aguilar, E., Dieguez, C., Millar, R.P., Tena-Sempere, M., 2012. Kisspeptins and reproduction: physiological roles and regulatory mechanisms. Physiological Reviews 92, 1235-1316. https://doi.org/10.1152/physrev.00037.2010.

Pinto, P.H.N., Balaro, M.F.A., Souza-Fabjan, J.M.G., Ribeiro, L.D.S., Bragança, G.M., Leite, C.R., Arashiro, E.K.N., de Moraes Silva, K., Da Fonseca, J.F., Brandão, F.Z., 2018. Anti-Müllerian hormone and antral follicle count are more effective for selecting ewes with good potential for in vivo embryo production than the presence of FecGE mutation or eCG pre-selection tests. Theriogenology 113, 146-152. https://doi.org/10.1016/j.theriogenology.2018.02.018.

Quignon, C., Beymer, M., Gauthier, K., Gauer, F., Simonneaux, V., 2020. Thyroid hormone receptors are required for the melatonin-dependent control of Rfrp gene expression in mice. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology 34, 12072-12082. https://doi.org/10.1096/fj.202000961R.

Quillet, R., Ayachi, S., Bihel, F., Elhabazi, K., Ilien, B., Simonin, F., 2016. RF-amide neuropeptides and their receptors in mammals: pharmacological properties, drug development and main physiological functions. Pharmacology & Therapeutics 160, 84-132. https://doi.org/10.1016/j.pharmthera.2016.02.005.

Revel, F.G., Saboureau, M., Masson-Pévet, M., Pévet, P., Mikkelsen, J.D., Simonneaux, V., 2006. Kisspeptin mediates the photoperiodic control of reproduction in hamsters. Current Biology: CB 16, 1730-1735. https://doi.org/10.1016/j.cub.2006.07.025.

Ruan, J., Xu, J., Chen-Tsai, R.Y., Li, K., 2017. Genome editing in livestock: are we ready for a revolution in animal breeding industry? Transgenic Research 26, 715-726. https://doi.org/10.1007/s11248-017-0049-7.

Schmidt, S.M., Belisle, M., Frommer, W.B., 2020. The evolving landscape around genome editing in agriculture: many countries have exempted or move to

exempt forms of genome editing from GMO regulation of crop plants. EMBO Reports 21, e50680. https://doi.org/10.15252/embr.202050680.

Simonneaux, V., 2020. A Kiss to drive rhythms in reproduction. The European Journal of Neuroscience 51, 509-530. https://doi.org/10.1111/ejn.14287.

Smith, J.T., Coolen, L.M., Kriegsfeld, L.J., Sari, I.P., Jaafarzadehshirazi, M.R., Maltby, M., Bateman, K., Goodman, R.L., Tilbrook, A.J., Ubuka, T., Bentley, G.E., Clarke, I.J., Lehman, M.N., 2008. Variation in kisspeptin and RFamide-related peptide (RFRP) expression and terminal connections to gonadotropin-releasing hormone neurons in the brain: a novel medium for seasonal breeding in the sheep. Endocrinology 149, 5770-5782. https://doi.org/10.1210/en.2008-0581.

Tan, W., Proudfoot, C., Lillico, S.G., Whitelaw, C.B.A., 2016. Gene targeting, genome editing: from Dolly to editors. Transgenic Research 25, 273-287. https://doi.org/10.1007/s11248-016-9932-x.

Tervit, H.R., Whittingham, D.G., Rowson, L.E., 1972. Successful culture in vitro of sheep and cattle ova. Journal of Reproduction and Fertility 30, 493-497. https://doi.org/10.1530/jrf.0.0300493.

Thorson, J.F., Prezotto, L.D., Cardoso, R.C., Sharpton, S.M., Edwards, J.F., Welsh, T.H., Riggs, P.K., Caraty, A., Amstalden, M., Williams, G.L., 2014. Hypothalamic distribution, adenohypophyseal receptor expression, and ligand functionality of RFamide-related peptide 3 in the mare during the breeding and nonbreeding seasons. Biology of Reproduction 90, 28. https://doi.org/10.1095/biolreprod.113.112185.

Thorson, J.F., Desaulniers, A.T., Lee, C., White, B.R., Ford, J.J., Lents, C.A., 2015. The role of RFamide-related peptide 3 (RFRP3) in regulation of the neuroendocrine reproductive and growth axes of the boar. Animal Reproduction Science 159, 60-65. https://doi.org/10.1016/j.anireprosci.2015.05.013.

Tsutsui, K., Ubuka, T., Son, Y.L., Bentley, G.E., Kriegsfeld, L.J., 2015. Contribution of GnIH research to the progress of reproductive neuroendocrinology. Frontiers in Endocrinology 6, 179. https://doi.org/10.3389/fendo.2015.00179.

Ubuka, T., Inoue, K., Fukuda, Y., Mizuno, T., Ukena, K., Kriegsfeld, L.J., Tsutsui, K., 2012. Identification, expression, and physiological functions of Siberian hamster gonadotropin-inhibitory hormone. Endocrinology 153, 373-385. https://doi.org/10.1210/en.2011-1110.