Integration of MOET and CRISPR/Cas9 RNP electroporation for MSTN genome editing in caprine zygotes

Abstract

Myostatin (MSTN) is a key negative regulator of skeletal muscle growth and an important candidate gene for genetic improvement of economically relevant livestock traits. CRISPR/Cas9-mediated genome editing using ribonucleoprotein (RNP) delivery offers a precise approach for targeted MSTN modification in caprine embryos. The present study aimed to establish a CRISPR/Cas9 genome-editing platform in MOET-derived caprine presumptive zygotes using electroporation. Presumptive zygotes were recovered following MOET from eight donor does, yielding 78 zygotes. All recovered zygotes were electroporated with a preassembled CRISPR/Cas9 RNP complex under optimized conditions (25 V, three square-wave pulses of 3 ms duration at 100 ms intervals). Following electroporation, embryos were cultured in vitro to assess developmental competence. All embryos developed to the morula stage, with two progressed to the blastocyst stage. During culture, four-cell embryos (n = 4) were selected for transfer into a synchronized recipient doe, while four morula-stage embryos were individually subjected to molecular analysis. Following whole-genome amplification and PCR, T7 Endonuclease I (T7E1) analysis detected cleavage at the targeted MSTN locus in three of four morulae (75%), indicating CRISPR/Cas9-associated modification. The four transferred embryos established pregnancy, and two fetuses were recovered on Day 57 of gestation. Both appeared morphologically normal, and T7E1 analysis detected targeted-locus cleavage in Fetus 1, whereas Fetus 2 showed no detectable cleavage. These findings demonstrate the feasibility of integrating MOET, CRISPR/Cas9 RNP electroporation, embryo culture, and zygote transfer as a practical platform for targeted genome modification in goats. Keywords: CRISPR/Cas9, Myostatin, MOET, Electroporation, Caprine genome editing