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Eye development occurs during the early stages of embryonic development in a highly complex and coordinated manner. This process is controlled by an evolutionary conserved molecular network that, if disrupted, results in severe congenital eye disorders. Identifying and studying genes involved in critical stages of early eye development may provide valuable insights into the molecular mechanisms driving the most severe ocular malformations. In this thesis, I used in vitro approaches to study the early stages of eye development to understand the molecular basis of eye malformations. I investigated the genetic cause of anophthalmia in the Ie mouse using long-read sequencing and OV organoids. I established an efficient CRISPR-Cas9 genome editing strategy to fluorescently tag an endogenous eye field transcription factor (EFTF) in mouse embryonic stem cells (mESCs). Moreover, I determined the effect of protein tagging in OV organoid morphology and gene expression. Analyses using short-read and long-read whole-genome sequencing (WGS) helped exclude a coding variation found in the critical interval in chrX and a structural variation as potential causes of anophthalmia in the Ie mouse. These results suggested that the cause of the severe eye malformation in this mouse is non-coding. Further experiments using optic vesicle (OV) organoids generated from Ie mESCs and mRNAseq did not show any evident gene expression dysregulation in this mouse. Therefore, more studies are needed to identify the molecular basis of anophthalmia in this mouse. Using different CRISPR-Cas9 genome editing strategies, I determined that Homology Independent Targeted Integration (HITI) was the most efficient way to generate a homozygous Pax6Halo/Halo mESC line. The functionality of Pax6 in Pax6Halo/Halo mESCs was assessed by its potential to develop eye-like structures in OV organoids. My results suggest that HaloTag knock-in lead to a partial loss of Pax6 function and, consequently, a mutant phenotype. Finally, the phenotype observed in OV organoids derived from Pax6Halo/Halo mESCs seems to be influenced by the culture conditions. Here, Pax6Halo/Halo organoids grown in Knockout Serum Replacement containing media (KSR) vs Chemically Defined Medium (CDM) were compared. A differential gene expression analysis identified genes involved in eye development and neurogenesis, which were upregulated in organoids grown in KSR and downregulated in CDM. These results suggest that KSR conditions promote organoid development towards mixed cell lineages rather than only eye development when there is a partial loss of Pax6 function.