P-794 Unraveling the function of GATA2 and GATA3 in mouse blastocyst formation via CRISPR/Cas9 gene editing

Abstract

Abstract Study question What is the effect of the CRISPR/Cas9 mediated knock-out (KO) of Gata2 and Gata3 on mouse preimplantation development/blastocyst formation? Summary answer Efficient generation of Gata3/Gata2 KO mouse embryos was achieved. Gata3 and Gata2 KO embryos could form blastocysts, suggesting a possible dispensability for blastocyst formation. What is known already GATA2 and GATA3 are important trophectoderm markers in mouse and human embryos. Prior studies, involving RNAi knockdown or conditional KO of Gata3, demonstrated high rates of morula arrest of Gata3-/- embryos, indicating its essential role in blastocyst formation. However, conflicting results showed embryonic lethality in Gata3-/- embryos only at 11-12 dpf. Besides, conditional KO of Gata2 suggested its dispensability in blastocyst formation, as Gata2-/- embryos could form blastocysts. It is of interest to validate these findings and potential functional redundancy between GATA2 and GATA3 in mouse embryos, by generating CRISPR/Cas9-mediated single and double KOs of Gata2 and Gata3. Study design, size, duration Upon assessment of the on-/ off-target effects of CRISPR/Cas9 components in mouse embryonic stem cells (mESC), CRISPR/Cas9 ribonucleoprotein (RNP) complexes were electroporated in mouse zygotes. Suitable control groups (non-targeted and scrambled targeted (inactive crRNA)) were incorporated. Embryos were genotyped by next generation sequencing (NGS), to assess the effect of gene KO on embryo morphology. Additionally, the expression pattern of Gata2 and Gata3 was examined in wild type and CRISPR/Cas9 targeted embryos, with immunofluorescent staining (IFS). Participants/materials, setting, methods Following CRISPR/Cas9 mediated targeting of Gata2 and Gata3, mouse embryos were cultured and morphologically monitored until day 4.5, alongside relevant control groups (scrambled, media control). Subsequently, DNA was extracted, and embryos were genotyped by PCR amplification of the region surrounding the target site, followed by NGS. IFS of mouse embryos at different developmental stages (E2.5, E3.0, E3.5 and E4.5) was performed to assess expression patterns and localization of both Gata2 and Gata3 throughout development. Main results and the role of chance Following the design of two gRNAs targeting either Gata2 or Gata3, on-target editing efficiency was tested in mESCs. The selected Gata2-targeting design resulted in a frameshift efficiency of 75%, whereas the Gata3-targeting design resulted in a frameshift efficiency of 78%. No off-target effects were observed in Gata2 and Gata3 targeted mESCs when examining in silico predicted off-target sites through PCR and NGS. Furthermore, 51% of Gata3 targeted and 41% of Gata2 targeted mouse embryos contained frameshift mutations in all cells. Notably, the majority of Gata3-/- (93%, n = 22) and Gata2-/- (94%, n = 16) embryos developed to blastocysts, similar to media control and scrambled groups. No statistically significant difference between Gata3-/- (n = 24, p = 0.326) and Gata2-/- (n = 17, p = 0.978) embryo morphology and scrambled (n = 96) embryo morphology was detected. IFS of control embryos (n = 16) showed that nuclear expression of GATA2 precedes nuclear expression of GATA3, indicating that GATA2 might control the expression of Gata3. Following IFS of Gata3 targeted embryos (n = 4), no nuclear signal for both GATA2 and GATA3 was observed, indicating that GATA3 could regulate the expression of Gata2. Furthermore, we created 100% double KO mouse embryos for Gata2 and Gata3 in two ways, enabling comprehensive exploration of functional redundancy in future studies. Limitations, reasons for caution Mosaicism (>1 genotype present in one embryo), off-target effects and chromosomal arrangements pose challenges regarding CRISPR/Cas9 germline genome editing. Zygote-stage delivery of RNP complexes is used to address mosaicism, and off-target effects are screened in mESCs via NGS. Future efforts aim to increase the sample sizes for stronger support. Wider implications of the findings Our goal is to identify the molecular pathways underlying unsuccessful implantation in mouse and human embryos. Utilizing CRISPR/Cas9 to generate KO embryos enhances our understanding of key molecular mechanisms, potentially shedding light on pregnancy failure due to failure of trophectoderm specification, formation or maintenance. Trial registration number not applicable