PROJECT SUMMARY/ABSTRACT Humans have evolved an expanded and elaborated brain capable of higher-order cognition. However, the sequence variants and resulting neural specializations that distinguish humans from other mammals are commonly dysregulated in diseases such as autism spectrum disorder (ASD). This suggests that human-specific non-coding variants are enriched for neural functions and may underlie genetic and phenotypic disease vulnerabilities. Thus, there is a critical need to identify and characterize the non-coding variants that underlie human neural specializations. However, identifying the causal variants that contribute to human neural specializations is a daunting challenge that has been likened to searching for needles in a haystack. In this proposal, Dr. Janet Song will improve prioritization of human-specific variants for further functional analysis using two complementary approaches (Aims 1 and 2) and determine whether prioritized variants regulate nearby gene expression in a high-throughput manner (Aim 3). Dr. Song will use the human-chimpanzee tetraploid system to link regulatory regions that are differentially accessible between species to nearby differential genes in neural progenitor cells and excitatory neurons, two cell types that are profoundly changed in humans and are commonly dysregulated in neurological diseases (Aim 1 – K99 phase). As a complementary approach, Dr. Song will identify constrained human-specific insertions and assess their contribution to ASD risk (Aim 2 – K99/R00 phase). Dr. Song will then evaluate the effects of human-specific variants on nearby gene expression in neural cell types using CRISPR inhibition screens (Aim 3 – K99/R00 phase). This K99/R00 proposal will support Dr. Janet Song in her pursuit of the genetic basis of human neural specializations and allow her to acquire new skills in comparative and functional genomics that will open up innovative approaches to explore this problem. This proposal will be initiated during the mentored period in Dr. Christopher Walsh’s lab at Boston Children’s Hospital / Harvard Medical School and continue in Dr. Song’s own lab upon securing an independent position. In addition to providing immediate insights into the genetic basis of human neural specializations, the proposed research will lay the foundation for Dr. Song’s independent research program. It will provide a framework for future studies in additional cell types or paradigms, pinpoint high-priority loci for single-locus studies, and identify a corpus of human-evolved elements that may contribute to genetic risk for neurodevelopmental and neuropsychiatric diseases. Long-term, Dr. Song’s independent research program will dissect how sequences that changed in humans relative to other mammals result in human-specific neural phenotypes, and ultimately, contribute to neurodevelopmental and neuropsychiatric diseases.