P-794 Cadherin1 is essential for blastulation: a CRISPR-Cas9 knock-out approach in human embryos

Abstract

Abstract Study question Is Cadherin1 required for human embryo blastulation? Summary answer Knock-out of Cadherin1 by Crispr/Cas9 technology in human embryos impairs cavitation and blastula stability. What is known already Embryo compaction involves an increase in intracellular adhesion mediated by E-cadherin. Concomitantly, the outer blastomeres undergo apical-basal polarization and are fated to generate the trophectoderm, the first epithelium of the embryo. Mice embryos devoid of E-Cadherin can complete compaction driven by maternal E-cadherin but fail to form a trophectodermal epithelium and a blastocoel. While mouse and human preimplantation development share common landmark events, there are also significant species-specific differences. To determine the role of Cadherin1 (CDH1) in preimplantation development, the E-Cadherin gene was targeted using the Crisper-Cas9 system in human 3PN embryos. Study design, size, duration This is a prospective basic research study; 64 tripronuclear zygotes (3PN) from patients undergoing IVF were collected between October 2018 and October 2019. 3PN zygotes were vitrified with pronuclei still visible, stored and warmed before processing. Participants/materials, setting, methods 58 3PN zygotes survived warming and were injected either with an equimolar combination of 3 guides targeting exon 2 of CDH1 (200ng/ul) or a scrambled control guide, along with Cas9; 3PN development was monitored by time-lapse microscopy, taking time of ICSI as T = 0. Culture was stopped at D6 or when embryos arrested for 24h. Genomic DNA was obtained by Multiple Displacement Amplification. Amplicon sequencing of on- and off-targets was performed to evaluate targeting efficiency. Main results and the role of chance 23 control and 29 treated 3PN embryos were successfully injected. In the control and treated group respectively, 10/23 (43.5%) and 15/29 (51.7%) embryos did not develop beyond the 8-cell stage; 1/23 (4.3%) and 3/29 (10.3%) embryos did not develop beyond the 16-cell stage; 4/23 (17.4%) and 3/29 (10.3%) embryos started to compact but failed to initiate cavitation. 8/23 (34.8%) and 8/29 (22.8%) started to cavitate (all differences non-significant, exact Fisher test). Interestingly, while 6/23 (26.1%) control embryos formed stable blastocysts, only 1/29 (3.4%) reached the stable blastocyst stage after CDH1 ablation (p = 0.035, exact Fisher test). To determine editing efficiency, we sequenced both the CDH1 exon2 and 7 off-target sites for each of the 3 guides used, in 6 control and 26 treated embryos. 14/26 (53.8%) of the treated embryos had severe disruptions, in CDH1 exon2, presenting up to 3 deletions and short indels between and around the guide sites in exon2, while 13/26 (46.2%) treated embryos were unaffected. Off-target sequences were unaffected in both groups. None of the edited embryos reached the blastocyst stage. Thus, loss of CDH1 compromises cavitation in developing human embryos, presumably by affecting cell-cell junctions and integrity of trophoblast cells, resulting in lower blastocyst rate formation. Limitations, reasons for caution Embryos analyzed in the study arise from 3PN embryos; the observed phenotype may be partially due to chromosomal abnormalities, although the difference in frequency of blastocyst formation between control and treated groups suggests a small effect of the sample type on the observed CDH1 knock-out phenotype. Wider implications of the findings We show that Cadherin 1 is necessary to reach a stable blastocyst stage in human preimplantation development, even though compaction and initial blastulation are possible. Further, our results confirm that 3PN embryos can be a useful model for testing gene function of candidate genes in human preimplantation development. Trial registration number NA