Efficient gene editing of BMP15, GDF9, and MSTN-but not the imprinted CLPG gene-in goat embryos via electrotransfection and handmade cloning

Abstract

CRISPR/Cas9 technology represents a powerful tool for advancing livestock breeding by enabling precise, on-target gene edits without the genomic mixing associated with traditional introgression methods. In this study, we employed a dual gRNA-based CRISPR/Cas9 strategy to induce targeted deletions and indel mutations in both reproductive and growth-related genes. These included the metacentric genes bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are associated with increased ovulation rate and litter size, as well as the telomeric genes myostatin (MSTN) and callipyge (CLPG), which are linked to muscle development and enhanced meat production. We employed an optimized electrotransfection protocol consisting of 10-20 µg of each plasmid DNA, 250 µL OptiMEM-GlutaMAX, and one million goat fibroblast cells. The electroporation was performed using a Bio-Rad system in a 4-mm cuvette, with two 10-millisecond pulses at 270 volts, separated by a 10-second interval. This protocol enabled efficient genome editing of goat embryonic fibroblast cells, which were subsequently used to generate cloned embryos via handmade somatic cell nuclear transfer (SCNT), involving manual enucleation and cell-oocyte fusion steps. Sequencing revealed high mutation rates (78-97%) and a predominance of biallelic edits in BMP15, GDF9, and MSTN. Notably, MSTN gRNAs with a 7-bp overlapping sequence at their 3' ends showed a high editing efficiency. In contrast, the imprinted CLPG gene exhibited a significantly lower mutation rate (~ 30%), likely due to epigenetic constraints. While overall mutation rates did not differ significantly between metacentric and telomeric genes, on-target deletions were more frequent in metacentric genes (43%) than in telomeric ones (20%). Embryo development rates from gene-edited cells were comparable to those from non-edited controls. These findings underscore the utility of combining electrotransfection with SCNT for efficient editing of non-imprinted genes and highlight the need for improved strategies to overcome barriers in editing imprinted loci.

Keywords: CRISPR/Cas9; Electrotransfection; Gene editing; Handmade cloning; Imprinted genes; Somatic cell nuclear transfer (SCNT).