PROJECT SUMMARY/ABSTRACT The importance of 3D genome structure on the evolution of human specific gene regulation, phenotypes, and speciation remains largely unknown. Here, I plan to characterize in a high- throughput manner the impact of topologically associated domains (TADs), which are genomic neighborhoods of self-interacting DNA, and CCCTC binding factor (CTCF) binding motifs, which help facilitate DNA looping, on cell viability and evolution. I hypothesize that a subset of TAD boundaries are critical for maintaining important gene regulation throughout evolution and that derived CTCF binding sites in the human lineage may have led to gene expression changes. I will interrogate this hypothesis by first performing a high-throughput CRISPR-deletion screen in which I will delete a set of over 300 evolutionarily conserved and human specific TAD boundaries in a human haploid cell line to determine if any of these boundaries are essential for cell viability (Aim 1). Second, I will identify human specific CTCF binding motifs that are gained or lost in humans or have orientation changes by comparing genome sequences from human, chimpanzee, and two extinct archaic hominids: Neanderthal and Denisovan. I will further interrogate the importance of a subset of these novel CTCF sites by CRISPR/Cas9 editing human cells to mimic that of an extinct genome and phenotype them for changes in gene expression changes and chromatin conformation. It will then be possible to determine if these changes caused differences in gene regulation and genome looping (Aim 2). Combined, this proposal will take a crucial step in further understanding how 3D chromatin structure can affect phenotypes especially as it relates to human evolution.