UCSF

The enumeration of human-specific genetic variants by comparative studies of ape genomes paired with advances in programmable genetic editing enables the functional interrogation of our evolutionary history. Because it is difficult to predict how millions of genomic alterations within the hominid lineage contribute to differences in phenotypes, there is a critical need for high-throughput, systematic approaches to probe genetic variation. Here, we develop a quantitative genome-scale platform for identifying the phenotypic consequences of human-specific genetic variants at a cellular and molecular level. First, we investigate the ancestral function of structural variant-sized human-specific deletions (hDels) by performing CRISPR interference (CRISPRi) genetic screens in chimpanzee induced pluripotent stem (iPS) cells. We further characterize the epigenetic state of chromatin at hDels using Omni ATAC-seq and CUT&Tag and perform single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes. We discover hDels removing cis-regulatory elements controlling the expression of proliferation-modifying genes including MBD3, MRPS14, and RPL26, and identify the cis-regulatory target genes of 16 nonessential hDels intersecting Omni ATAC-seq, H3K4me1, and H3K27ac. Separately, we investigate the evolution of gene regulatory networks during recent hominid evolution by performing paired CRISPRi genetic screens in human and chimpanzee iPS cells. We identify 75 genes with species-specific effects on cellular proliferation, many of which interact in biological pathways including RNA metabolism, chromatin organization, cell cycle phase transition, and UFMylation. Together, these findings underscore the importance of deletions as a source of evolutionary innovation and suggest that the regulation of essential cellular processes has evolved along the human lineage.