Cracking the role of RFRP3 in seasonal breeding with genome editing. – GMO-Phen

1/ Use the CRISPR-Cas9 methodology to invalidate the Npvf gene, which codes for the RFRP3 peptide (involved in seasonal reproduction), in sheep and hamsters, two species with seasonal reproduction (short-day and long-day breeders, respectively).
2/ Perform an endocrine/behavioural phenotyping of the animals thus generated in order to test the impact of the deletion on the seasonality of reproduction.
3/ Develop and use the single cell RNAseq (scRNAseq) methodology to obtain a fine molecular characterization of Npvf neurons in sheep and hamsters, with the working hypothesis that a difference in neurotransmitter (activator/inhibitor) could explain the phenotypes in these species, which display opposite seasonal patterns of reproduction (also called phenology).

1/ The generation of transgenic sheep was planned over two consecutive breeding seasons: 2021-2022 and 2022-2023. This involved carrying out 5 similar experiments each year, each involving 6 sheep donors of fertilized oocytes (super-ovulation protocol) and 6 ewes receiving fertilized oocytes edited by CRISPR-Cas9 (from 1 to 3 reimplanted oocytes/sheep ). Year 1 experiments went according to plan, between September 2021 and February 2022. Of the 30 recipient ewes, 6 were pregnant (in 4 different experiments) giving birth to 7 lambs (one twin birth). 3 of these lambs – 1 male and 2 females; founders – carry at least one edited Npvf allele. This result is encouraging and demonstrates that our approach is functional. We have already started the year 2 experiments by modifying the gRNA approach (1 single guide instead of 2) and we hope to obtain a male whose two alleles would be edited, in order to be able to establish an ovine Npvf-KO line. The animals already obtained should provide some preliminary answers. Concerning hamsters, the methodology proves to be more difficult to implement than expected and despite the various attempts, no gestation (of unedited oocytes) has been obtained although our sampling and culture methods allow us to to obtain young blastocysts. Developments are continuing and a change in the methodological approach (electroporation in vivo, so-called GONAD method already used in mice and rats) is under discussion.
2/ Phenotyping edited animals requires HAVING the edited animals – so this part of the project is still in progress.
3/ A pilot scRNAseq manipulation was carried out in sheep between June and October 2022. This experience was rich in lessons, partly compatible with those of the literature: there are few neurons in our structure of interest and their structure (long extensions, axons and dendrites) makes them very fragile to the dissociation process. The scRNAseq result is quite valuableas it demonstrates our ability to do the procedure, but also clearly indicates that only glial cells are present in our sample. We therefore decided to switch to single nuclei RNAseq (snRNAseq) which should make it possible to remove this limitation. The updates took place between February and June 2022 and the 1st complete manipulation is planned for the end of November. If this methodology proves profitable, it will be applied to samples from hamsters in 2023.

The methodological adaptations should allow us to obtain other sheep with editing of the Npvf gene, necessary to establish a line (outside the time frame of this project). The development of snRNAseq instead of scRNAseq should give us access to the seasonal transcriptome of these neurons in sheep and hamsters, which will allow us to directly test our hypothesis of an opposite neurochemical phenotype between sheep and hamsters.

Publications in peer-reviewed journals thanks to the support of the ANR:

1. Dardente & Simonneaux. Journal of Neuroendocrinology, 34 (4): e13124, 2022. doi: 10.1111/jne.13124
2. Dardente et al. Journal of Neuroendocrinology, 34 (10): e13198, 2022. doi: 10.1111/jne.13198
3. Angelopoulou E, Kalsbeek A, Simonneaux V. Age-dependent change of RFRP-3neuron numbers and innervation in female mice. Neuropeptides. 92: 102224. 2022 doi: 10.1016/
4. Angelopoulou E, Inquimbert P, Klosen P, Anderson G, Kalsbeek A, Simonneaux V. ;Daily and Estral Regulation of RFRP-3 Neurons in the Female Mice. J Circadian Rhythms 19:4. 2021. doi: 10.5334/jcr.212.

Most species exhibit seasonal breeding as an adaptive response to environmental changes. However, seasonal species mate either under short (e.g. sheep) or long days (e.g. hamster). We highlighted roles for the mammalian neuropeptides RFRP3 (RF-amide Related Peptide 3 encoded by the Npvf gene) and KISS1 (Kisspeptin), produced by hypothalamic neurons. In contrast to KISS1, the role of RFRP3 in seasonal breeding is unclear. We hypothesize that integration primarily occurs at the level of Npvf neurons, which then dictate species-specific seasonal breeding. The current project will investigate this in Syrian hamster and sheep. The aims are to provide evidence for the implication of the Npvf gene through CRISPR-Cas9 invalidation and to characterize RFRP3-producing neurons with respect to neurotransmitter expression and sex-steroid feedback using scRNAseq and to explore neuronal wiring between KISS1 and RFRP3 populations, as plausible substrates for opposite seasonal phenologies