CRISPR/Cas9-Mediated Introduction of the R353Q SLC25A12 Mutation in Ovine Granulosa Cells and Production of Edited Blastocysts via Somatic Cell Nuclear Transfer

[Purpose] Mitochondrial disorders caused by mutations in genes involved in energy metabolism often lead to severe neurological impairment. Among them, the R353Q point mutation in the solute carrier family 25 member 12 (SLC25A12) gene, encoding the mitochondrial aspartate–glutamate carrier 1 (AGC1), is associated with epileptic encephalopathy, hypomyelination, and autism spectrum features in humans[1,2]. To date, no large-animal model reliably reproduces the chronic neurological phenotype associated with this mutation. The aim of this study was to generate an ovine cellular and embryonic model carrying the R353Q mutation through CRISPR/Cas9 knock-in editing of granulosa cells (GCs) followed by somatic cell nuclear transfer (SCNT) to obtain blastocyststage embryos. [Method] A CRISPR/Cas9 ribonucleoprotein (RNP) complex and a singlestranded oligonucleotide donor (ssODN) were designed based on protein and nucleotide alignments between human and sheep AGC1. Because the ovine mutation requires substitution of two nucleotides within the target codon, bioinformatic tools (CRISPOR and Cas-OFFinder) were used to select a high-fidelity gRNA positioned optimally for HomologyDirected Repair (HDR) and to minimize off-target activity. The ssODN included symmetric homology arms and silent mutations to prevent re-cutting by Cas9.GCs were collected from sheep ovaries obtained at local abattoirs. After in-vitro expansion, GCs were transfected with the CRISPR/Cas9 RNP–ssODN construct using a lipid-based system (Lipofectamine). Molecular validation of targeted knock-in GCs is currently in progress. Transfected GCs were sent to University of Teramo (Italy) where they were subsequently reprogrammed and used as nuclear donors for SCNT[3]. In vitro matured oocytes were enucleated, reconstructed with the transfected donor nuclei, fused, activated, and cultured to the blastocyst stage. [Results] Edited GCs maintained control-like morphology and proliferation. SCNT using the transfected and reprogrammed donor GCs resulted in the successful production of blastocyststage embryos, all of which were cryopreserved. Molecular analyses of both donor cells and derived blastocysts are underway to assess the presence and accuracy of the intended R353Q knock-in event. [Discussion] The workflow combining CRISPR/Cas9-mediated editing of GCs with SCNT proved effective for producing ovine cloned embryos derived from genome-edited donor cells, even before completion of molecular validation. This approach provides a functional platform for evaluating HDR efficiency in a large-animal model. [Conclusion] This study establishes the feasibility of generating ovine blastocysts from CRISPR-transfected GCs via SCNT. Ongoing validation of the R353Q mutation will determine whether the resulting embryos represent the first step toward establishing a large-animal model of AGC1 deficiency, with potential applications in the study of mitochondrial and neurometabolic diseases.