Uncovering the developmental defects associated to Holstein haplotype 3 by CRISPR technology

Pérez-Gómez A., Álvarez-Sala M., Inglés-Pedreño Á., Lamas-Toranzo I., Galiano-Cogolludo B., Gabriela Hamze J., ...More

REPRODUCTION, FERTILITY AND DEVELOPMENT, vol.35, no.2, pp.205, 2022 (SCI-Expanded)

  • Publication Type: Article / Article
  • Volume: 35 Issue: 2
  • Publication Date: 2022
  • Doi Number: 10.1071/rdv35n2ab162
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, EMBASE, MEDLINE, Veterinary Science Database
  • Page Numbers: pp.205
  • Akdeniz University Affiliated: Yes


Holstein haplotype 3 (HH3) is a lethal allele present in the Holstein population that causes pre-term mortality in double-carrier (homozygous) embryos. The stage at which homozygous embryos arrest their development remains unknown, and this information is critical to evaluate the economic losses associated with the inadvertent cross between HH3 carriers and, thereby, to estimate the cost-benefit balance of genotyping heifers. The lethality associated with HH3 is mediated by a single nucleotide polymorphism (SNP) causing a nonsynonymous change (T/C) in exon 24 of the structural maintenance of chromosomes 2 (SMC2) gene. The objective of this study has been to elucidate the developmental impact of SMC2 ablation on early development. For that purpose, we have employed CRISPR technology in wild-type (WT) embryos to generate frame-shift mutations in the exon of SMC2 affected by HH3 haplotype. In vitro-matured bovine oocytes were denuded and divided into two groups: one was microinjected with Cas9-encoding mRNA (group C, serving as injection control), and the other with Cas9-encoding mRNA at 300 ng/µL (C) and guide RNA at 100 ng/µL (G) directed to exon 24 of SMC2 (group C + G). Group C is solely composed of WT embryos and group C + G may contain WT (unedited) or edited embryos; the latter being knockout (KO) when they only harbour frame-disrupting alleles. Following microinjection, in vitro fertilisation (IVF) was performed, and zygotes were cultured in SOF medium until Day 8, when blastocysts were fixed for immunohistochemistry (IHC) to distinguish inner cell mass (ICM, SOX2+) and trophectoderm cells (CDX2+). Following IHC and fluorescence structured-illumination analysis, embryos from group C + G were genotyped by miSeq to identify KO (containing only frame-disrupting alleles unable to generate functional SMC2) and edited in-frame (containing at least one in-frame allele which may produce a functional version of SMC2) embryos. No significant differences were found in blastocyst rates between the two microinjection groups (35.9 ± 3.6 vs 27.4 ± 3.8%, mean ± s.e.m., for group C and group C + G, respectively; t-test P > 0.05). 39/39 embryos analysed in group C + G contained edited alleles, and nine of them contained only frame-disrupting alleles (KO embryos). IHC was conducted in 21 WT (from group C), 30 edited in-frame, and 9 KO embryos. Total (DAPI+) and trophectoderm (CDX2+) cell number was significantly higher in WT or edited in-frame embryos compared to KO (total: 112.8 ± 11.8 vs 85.3 ± 7.8 vs 48.1 ± 2.8; CDX2+: 90.4 ± 11 vs 62.8 ± 6.9 vs 32.3 ± 1.6 for WT, in-frame, and KO, respectively; ANOVA P < 0.05). The number of SOX2+ cells was significantly higher in WT embryos compared with KO embryos (29.6 ± 3.3 vs 21.9 ± 3.4 vs 7.4 ± 1.9 for WT, in-frame, and KO, respectively; ANOVA P < 0.05). In conclusion, SMC2 disruption at exon 24 causes cell proliferation defects already noticeable at the blastocyst stage, well before maternal recognition of pregnancy.