O-216 CRISPR/Cas9 mediated knock-out (KO) reveals a divergent role for trophectoderm markers GATA2/3 in the mouse and human preimplantation embryo
Authors: Cosemans G , Boel A , Bekaert B , Pascal E , Stamatiadis P , Stoop D , Chuva De Sousa Lopes S.M , Menten B , Coucke P , Lluis F , Heindryckx B 
Published: June 2022

Abstract

Study question
What is the effect of CRISPR/Cas9-mediated KO of trophectoderm (TE) markers GATA2/3 on embryo development and lineage commitment in both mouse and human preimplantation embryos?

Summary answer
CRISPR/Cas9-mediated KO of GATA2/3 points to interspecies differences in TE regulation and potential cross-talk between trophectoderm and inner cell mass (ICM) in mouse preimplantation embryos.

What is known already
GATA3 and its isoform GATA2 are major TE markers regulating the first lineage segregation, operating downstream of the HIPPO-pathway. Although recent evidence suggests that the HIPPO-pathway is conserved across mouse and human, it is unknown whether GATA2/3 share similar interspecies function during preimplantation development. GATA3 RNA depletion experiments in mouse embryos revealed a compensatory upregulation of GATA2, that potentially masked the observed phenotype. Upon double KO (DKO), the phenotype appeared more severe, as embryos were unable to cavitate. However, the precise effect of the (D)KO on embryo development was not investigated thoroughly, and should be expanded towards human preimplantation embryos.

Study design, size, duration
Mouse embryonic stem cells (mESCs), mouse zygotes and donated human spare oocytes were targeted. CRISPR/Cas9 ribonucleoprotein complexes, either targeting Gata3/GATA3, Gata2 or both, were delivered via nucleofection, electroporation or co-injected with sperm, in mESCs, mouse zygotes or human oocytes, respectively. Appropriate non-targeted control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing were applied to check for gene editing efficiency and the impact of KO on embryonic development.

Participants/materials, setting, methods
The targeted embryos and controls were cultured for 4.5 (mouse) or 6.5 days (human) in vitro. They were stained for different developmental markers, including TEAD4 and CDX2 (TE), OCT4 and SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE). Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency, while in silico predicted off-target sites were evaluated in targeted mESCs.

Main results and the role of chance
GATA3 KO mouse embryos exhibited morula arrest (94%; n = 16). All GATA3-edited mouse embryos exhibited a reduction of CDX2-positive cells. From the 12 full KO embryos, four showed a decreased number of NANOG-positive blastomeres. No effect was observed for TEAD4 and OCT4. Complete KO morulas were devoid of SOX2 expression.
GATA2 KO mouse embryos could still form blastocysts (19% morula arrest, n = 21 embryos), even when harboring 100% frameshift mutations. KO did not noticeably influence cell number nor the expression of GATA3 or NANOG.
GATA2/3 DKO mouse embryos could still form blastocysts (38% morula arrest, n = 21), showing a milder phenotype compared to GATA3 KO embryos. In the presumed DKO blastocysts, the whole ICM is NANOG-positive and increased in cell number. SOX2 expression was still retained in the ICM, but presumed polar TE was also SOX2-positive.
Three out of four GATA3 KO human embryos harboring 100% frameshift mutations, were surprisingly able to form blastocysts. In one embryo, no morphological TE could be formed, while the others showed one or two CDX2-positive cells in TE. No effect was observed for TEAD4 and OCT4. In addition, all KO embryos displayed an increase in ICM/TE fraction. Some outer cells in KO blastocysts were NANOG-positive.

Limitations, reasons for caution
CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mESCs. The observations of the study will be consolidated by increasing the sample size, especially in human.

Wider implications of the findings
Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the GATA transcription factor family could significantly improve our understanding of several pregnancy-related complications related to trophectoderm specification, such as early miscarriage or preeclampsia.

Trial registration number
NA