Construction of transgenic cell line for production of a canine model of Fragile X Syndrome

The aim of the present study is to establish a knockout (KO) cell line of FMR1, the gene responsible for Fragile X Syndrome (FXS), using CRISPR/Cas9 to create a canine model of autism. To construct a transgenic cell line, primary fibroblasts from the skin tissue of Beagle dogs were established, CRISPR/Cas9 vectors (dFXS, FMR1-gRNA1- gRNA2 -Cas9-EGFP) was constructed, the transfection conditions were optimized, fluorescence-expressing cells were separated via Fluorescence Activated Cell Sorting (FACS), and the insertion/deletion (inDel) of the mutation by CRISPR/Cas9 was verified. As a result, a dFXS vector of 11,875 bp was obtained, in which two single-guided RNAs (sgRNAs; gRNA1 and gRNA2) were inserted as targets for exon 1, where the start codon of FMR1 gene is located, using the most efficient transfection conditions(1700 V, 1 pulse, 20 widths). In addition, FACS showed that the dFXS vector was around 90% efficient in InDel formation by CRISPR/Cas9. In the future, the FMR1 KO transgenic cell line constructed in this study could be cloned using somatic cell nuclear transfer (SCNT) for the study of neurological diseases, including autism, and for the development of canine models of other human diseases for the development of novel drugs.