Human Accelerated Regions: Functional Studies and Methodological Approaches (A Review)

Human accelerated regions (HARs) are short genomic sequences that, despite being highly conserved across vertebrates, have undergone an unusually high rate of nucleotide substitutions in the human lineage. Although most HARs are located in non-coding regions, accumulating evidence suggests that many function as developmental regulatory elements, fine-tuning gene expression during fundamental neurodevelopmental processes such as radial glial cell proliferation, neurogenesis, cortical lamination, and others. Consequently, deciphering the functional consequences of human-specific sequence changes in HARs represents a major challenge in evolutionary genomics, as it holds the key to understanding the genetic basis of human uniqueness. In this review, we summarize recent advances in experimental approaches for dissecting HAR functions. We discuss how epigenomic profiling and chromatin conformation techniques pinpoint HARs with regulatory potential and connect them to target genes. We then detail functional validation strategies, ranging from classic in vitro and in vivo reporter systems to high-throughput screens like massively parallel reporter assays (MPRAs). Furthermore, we describe genome editing approaches, particularly CRISPR–Cas9, CRISPRi/a, and base editing, that enable causal testing of HAR functions in human cellular and animal models. The integration of these complementary methods provides a comprehensive framework for elucidating how specific sequence changes in HARs have reshaped gene regulatory networks during human evolution. We conclude by outlining current methodological challenges and future directions toward a systems-level understanding of HAR functions across cellular, developmental, and organismal contexts.