154 Gene editing via CRISPR/Cas9 of in vivo- and in vitro-derived bovine zygotes by electroporation

CRISPR/Cas9 has been used to efficiently edit the genomes of embryos in many animal models. It is relatively simple to derive large numbers of in vivo fertilized zygotes for gene editing experiments in small mammal models. However, harvesting in vivo fertilized zygotes in cattle is more complex. Here, we use a minimally invasive endoscopic method for harvesting in vivo fertilized zygotes by oviductal flushing of superovulated heifers, followed by subsequent electroporation of collected zygotes with CRISPR/Cas9 ribonucleoproteins (RNPs). Flushed presumptive zygotes were then stored in synthetic oviductal fluid (SOFaa) before electroporation. In addition, IVM oocytes (TCM-199 + 10% estrous cow serum, 38.5°C, 5% CO₂, 5% O₂) fertilized in vitro (Fert-TALP, 2 × 10⁶ sperm mL⁻¹, 38.5°C, 5% CO₂, 5% O₂) served as controls. With targeting of exon 1 of the tyrosinase (Tyr) gene, zygotes were electroporated in 1-mm gap cuvettes (Bio-Rad) in groups of ~20 in 20 μL of OptiMEM medium containing 3 μM Cas9 RNPs (IDT Cas9 to anti-Tyr guide RNA ratio = 1:1). Electroporation was performed in three replicates at three electrical potentials, namely 20, 25, and 30 V, using a Biojet CF 50. The other electroporation parameters were fixed at five repetitions of 2-ms square wave pulses at 100-ms intervals. After electroporation, presumptive zygotes were cultured under standard embryo culture conditions (SOFaa + 0.3% bovine serum albumin, 5% CO₂, 5% O₂, 38.5°C, humidified air). Embryo survival, cleavage, and developmental rates to the blastocyst stage until Day 9 were tracked. Subsequently, editing rates were verified with Sanger sequencing followed by sequence alignment and analysis using the online Inference of CRISPR Edits (ICE) tool (https://ice.synthego.com/#/). Statistical analysis of significance between groups was checked by pairwise one-way ANOVA using Sidak correction for multiple comparisons. After superstimulation of 21 heifers, 12 zygotes on average were flushed per animal. The results of the present study revealed that electroporation of in vivo-derived zygotes (n = 195) using 20 V (n = 61) yielded significantly higher survival (83.6% vs. 42.8% vs. 20.7%), cleavage (65.6% vs. 37.9% vs. 40.0%), and day-9 blastocyst rates (47.5% vs. 21.4% vs. 16.5%) compared with 25 V (n = 61) or 30 V (n = 73), respectively. In contrast, electroporation of in vivo-derived zygotes using 20 V resulted in significantly lower editing rates (9.7% vs. 61.1% vs. 50.0%) than 25 V and 30 V. Comparison of survival rates after electroporation (25 V, five repetitions of 2-ms square wave pulses) indicated higher survival of in vitro-derived (n = 22) compared with in vivo-derived (n = 61) zygotes (83.3% vs. 42.8%); whereas, editing rates of subsequent blastocysts following zygote electroporation of in vivo- and in vitro-derived zygotes did not reveal differences (61.1% vs. 75.0%). In contrast, subsequent blastocysts of in vitro-derived zygotes (n = 8) showed a significantly higher rate of editing mosaicism compared with those derived from in vivo zygotes (50.0% vs. 16.6%). These are the first confirmed gene-edited bovine embryos produced from in vivo fertilized zygotes to the best of our knowledge. This method could offer the option of using the zygotes of high-value cows or cows with known genotypes for genetic engineering in the future. Given that electroporated bovine zygotes can be transferred back to the oviduct endoscopically, our future attempts will focus on genome editing in bovine embryos developed almost completely in vivo.