5-methylcytosine (5mC) is a gene-regulatory mark predominantly associated with transcriptional repression when deposited at vertebrate CpG-rich genomic sequences. 5mC can be actively removed from the genome by TET enzymes that convert 5mC to 5-hydroxymethylcytosine (5hmC) eventually leading to its complete removal. It was recently demonstrated that during vertebrate embryogenesis, the majority of such active DNA demethylation processes occur on a subset of highly conserved CpG-island enhancers linked to key developmental pathways. Notably, such CpG-island enhancers form only a minor percentage of the total enhancer repertoire. To begin understanding how proteins with 5(h)mC binding capacity participate in the regulation of such enhancers, we combined CRISPR/Cas9 genome editing with reduced representation bisulfite sequencing (RRBS) in zebrafish embryos to assay the 5mC state of these sequences in zebrafish knockouts. Initial data reveal diverse transcription factor families, including HOX, SOX, MEIS family members, as well as RNA-binding (RBM) and paraspeckle (NONO, PSPC1) proteins as likely contributors to the formation of proper chromatin state of CpG-island enhancers. These results suggest a diverse range, and potential network, of transcription factors and RNA binding factors involved in the developmental demethylation of key enhancers related to body plan formation in vertebrates.