5-methylcytosine (5mC) is a gene-regulatory mark associated with transcriptional repression. 5mC can be erased through the catalytic action of Ten-eleven translocation (TET) methylcytosine dioxygenases (TET1, TET2, TET3), which oxidize 5mC resulting in its removal from the genome. In vertebrates, TET enzymes facilitate DNA demethylation of regulatory regions linked to genes involved in developmental processes. Consequently, TET ablation leads to severe morphological defects and developmental arrest. Here we describe a system that can facilitate the study of relationships between TET enzymes, 5mC, and embryo development. We provide detailed descriptions for the generation of F0 zebrafish tet1/2/3 knockouts using CRISPR/Cas9 technology and elaborate on the strategies to assess the impact of TET loss by reduced representation bisulfite sequencing (RRBS).TET family enzymes normally oxidize 5-methylcytosine (5mC) in DNA, and play critical roles in shaping the epigenome. Despite their importance, assessing TET activity can be difficult, particularly given the challenge of studying modifications to single nucleobases within complex DNA substrates. We recently demonstrated that in addition to acting on 5mC, TET enzymes can act promiscuously on unnatural nucleobases. Here, we describe how these alternative unnatural substrates can be employed in facile assays to detect and quantify TET activity. DNA containing unnatural 5-vinylcytosine (vC) can be used as a direct endpoint reporter of TET activity, a method that can potentially be adapted to high-throughput platforms. Complementarily, DNA containing unnatural 5-ethynylcytosine (eyC) can trap and inactivate TET enzymes upon reaction, a strategy that can be used to extract active TET enzymes from a complex cellular milieu. We present a detailed PCR-based protocol to synthesize DNA probes with either natural or unnatural modifications, and methods for using these probes to track TET activity either in vitro or in cell extracts.TET proteins are methylcytosine dioxygenases that interact directly with chromatin to shape the DNA methylation landscape. To increase the understanding of TET protein function in a specific cellular context, it is important to be able to map the interactions between TET proteins and DNA. This ChIP-seq protocol details our procedure to analyze TET2 bound DNA in disuccinimidyl glutarate (DSG) and formaldehyde-crosslinked chromatin but can also be adapted to study other TET enzymes.Ten-eleven Translocation (TET) enzymes are methylcytosine dioxygenases that are involved in multiple cellular processes, including cellular differentiation and forced cell fate conversions. However, deciphering the molecular mechanisms underlying epigenetic control exerted by these proteins has been hampered by technical limitations, which prevent the identification of essential partners that work in concert with these enzymes to modulate gene expression. In this chapter, we provide a comprehensive description of cutting-edge methods designed to assess physical interactions between sequence-specific transcription factors and the TET2 enzyme.The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. NVP-BEZ235 Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.In recent years, workflows coupling DNA affinity purifications from crude nuclear extracts with quantitative mass spectrometry-based proteomics have enabled comprehensive mapping of protein-DNA interactions in an unbiased manner. Here, we describe a detailed protocol for one such method in which affinity purifications with extracts from cells or tissues of interest are combined with a chemical stable isotope labeling method, dimethyl labeling, to identify specific interaction partners for (hydroxy)methylated and non-methylated DNA sequences of interest.Aberrant promoter hypermethylation leads to gene silencing and is associated with various pathologies including cancer and organ fibrosis. Active DNA demethylation is mediated by TET enzymes TET1, TET2, and TET3, which convert 5-methylcytosine to 5-hydroxymethylcytosine. Induction of gene-specific hydroxymethylation via CRISPR/Cas9 gene technology provides an opportunity to reactivate a single target gene silenced in pathological conditions. We utilized a spCas9 variant fused with TET3 catalytic domain to mediate gene-specific hydroxymethylation with subsequent gene reactivation which holds promise for gene therapy. Here, we present guidelines for gene-specific hydroxymethylation targeting with a specific focus on designing sgRNA and functional assessments in vitro.Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.