Histone variants play an additional role in epigenetic regulation, and dysregulation of histone variants has been shown to lead to a poor prognosis in diseases, such as cancer. MacroH2A, a non-canonical H2A variant, is unique because it is the largest of the histones, approximately 40 kDA. It consists of a histone folding region, a positively charged linker (which has been shown to compact chromatin in vitro), and a macrodomain. The three isoforms of macroH2A have been shown to play varied roles in the regulation of chromatin and gene expression, with macroH2A1.1 driving activity of some enhancers while macroH2A1.2 and macroH2A2 associating with gene repression at specific genes, as well as in heterochromatin and on the inactive X chromosome. In particular, macroH2A2 has been implicated in maintaining cell differentiation and preventing reprogramming. To study the function of macroH2A, we generated macroH2A knockouts in a stable diploid cell line (RPE-1 hTERT) using CRISPR/Cas9. Our results indicate that the loss of macroH2A1 does not affect transcription but does increase accessibility around promoters. Micro-C data also reveals that there are no significant changes in the global 3D structure, but upon macroH2A knockout, we observe localized changes in chromatin looping. Our ongoing work uses perturbations, including macroH2A overexpression, to further understand how macroH2A affects chromatin compaction and 3D organization of chromatin.