152 Evaluation of different transfection methods for the generation of gene-edited bovine embryos

CRISPR/Cas9-mediated genome editing in livestock has been applied for various purposes, such as improving food production and enhancing animal welfare and environmental sustainability. However, the lack of efficient approaches for the delivery of CRISPR molecules into zygotes has been a technical hurdle for high-throughput applications of gene editing in livestock. In this study, we aimed to evaluate different transfection methods for delivering CRISPR/Cas9 to zygotes to assist in establishing a simple and efficient workflow to generate bovine gene-edited embryos. We compared three different CRISPR delivery methods, including lipofectamine CRISPRMAX transfection (Thermo Fisher Scientific) and Neon and NEPA21 electroporation systems (Thermo Fisher Scientific and Bulldog Bio, respectively). For all transfections, we used the Bos taurus PRLR (prolactin receptor) gene as a target and designed two single-guide (sg)RNAs to delete 173 bp in exon 9 of the gene. The sgRNAs were complexed with Alt-R® S.p. HiFi Cas9 Nuclease V3 (Integrated DNA Technology) to form ribonucleoprotein (RNP) at a 1:1 molar ratio, which was then delivered into bovine Day 1 zygotes. By refining the procedure, we found that CRISPRMAX had minimal effect on embryo viability, as evidenced by the embryo cleavage rate (95.5%, n = 150) and hatching rate (33.6%), which were equivalent to those of the control groups (CRISPRMAX only and untreated). PCR genotyping using primers covering the target loci showed that 10 out of 45 CRISPRMAX transfected embryos (22.2%) possessed PRLR deletion. More PCR screening is ongoing. We evaluated different parameters for the Neon and NEPA21 electroporation systems. As expected, we noted that the higher the voltage and number of pulses, the lower the viability of the embryos. We tested voltages between 550 and 850 V for Neon electroporation, all using 10 ms and one pulse. So far, 750 V provided the best outcome, as it had relatively higher embryo cleavage (47.5%, n = 40) and hatching rate (12.5%) than using 850 V, and by PCR, we found 9 out of 72 embryos (12.5%) possessed PRLR deletions. We also optimized the parameters for the NEPA21 electroporation system. We found the parameters for poring (40 V, 3.5-ms wavelength, 50-ms pulse interval, four pulses, 10% decay [±pulse orientation]) and transfer (95 V, 50-ms wavelength, 50-ms pulse interval, five pulses, 40% decay [±pulse orientation]) had advantages in all the parameters we tested, based on the embryo cleavage (63.5%, n = 80) and hatching rate (19%). Further analysis of the gene editing efficiency and optimization of the three delivery methods are ongoing. We aim to define the parameters for a good balance between embryo viability and gene editing efficiency. The outcomes will provide valuable insights into enhancing the gene editing outcomes in bovines and promoting the accelerated and widespread application of gene editing technology in livestock.