Simultaneous CRISPR-on activation of TFAP2C and SMARCA4 improves developmental rates and increases trophoblast cells in bovine embryos

ALBERIO V; SAVY V; YAURI FELIPE M; RATNER LD; FERNANDEZ-MARTÍN R; SALAMONE DF

Galway

Simposio; The 11th International Ruminant Reproduction Symposium; 2023

bsas

Application: To produce trophoblast-enriched embryos to improve maternal-fetal recognition and implantation rates of in vitro-derived embryos.Introduction: Assisted reproductive techniques are widely used to produce bovine embryos for commercial or research purposes. In cattle, aberrant differentiation of trophoblast cells during embryo development leads to failures in pregnancy establishment and placentation. In this work, we sought to induce the simultaneous activation of TFAP2C and SMARCA4 expression by using CRSPR technology in bovine embryos to generate trophoblast-enriched blastocysts.Materials and Methods: Four non-overlapping single guide RNAs (sgRNAs) targeting TFAP2C and SMARCA4 were synthesized. Presumptive IVF zygotes were microinjected with a 9-μm pipette with a mix containing 100 ng/μl dCas9VP160 mRNA, 50 ng/μl of a mix of the 8 sgRNAs and 2 μl 10 % Polyvinylpyrrolidone (PVP) (TF-SM group). A group without sgRNAs (SHAM) and a non-injected group (IVF) served as controls. Embryos were cultured in synthetic oviductal fluid medium supplemented with 2.5% fetal calf serum at 38.5°C in humidified gas mixture (5% CO2, 5% O2, 90% N2) and developmental rates were determined (Fisher´s exact test). Samples were collected in pools of 10 embryos and 5 blastocysts at days 2, 4 and 7 of development to perform RTqPCR analysis (t test). Data were normalized to the IVF control. The expression of trophectoderm-specific marker CDX2 was analyzed in injected and SHAM blastocysts by immunostaining. Samples were scanned using an inverted confocal microscope and number of cells was determined by the ImageJ Software (t test). At least 3 biological replicates were included.Results: Embryo development was improved (p ≤ 0.05) in TF-SM (n=238) relative to the SHAM control (n=230) at cleavage (89.9 vs 83%), morulae (68.9 vs 48.6) and blastocyst (44.5 vs 30.8) stages. TFAP2C transcript abundance was significantly increased at days 2 (7-fold) and 4 (2.3-fold) in the TF-SM group relative to IVF control. However, SMARCA4 and CDX2 only showed significant differences at day 4 (2.3-fold and 2.2-fold, respectively) and GATA3 transcripts exhibit 1.8-fold difference (p ≤ 0.05) at day 2, between TF-SM and IVF control. No differences at the expression levels were observed at day 7. The number of CDX2+ cells (n=35 for both groups) in TF-SM group was statistically higher.Conclusion: The simultaneous activation of TFAP2C and SMARCA4 increased the transcripts levels of the analyzed genes until day 4 post-microinjection. A significant improvement in blastocyst development and in the proportion of trophectoderm cells were achieved compared to the SHAM control.