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A key challenge of the modern genomics era is developing data-driven representations of gene function. Here, we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-scale genotype-phenotype maps comprising >20,000 single-gene CRISPR-Cas9-based knockout experiments in >30 million cells. Our optical pooled cell profiling approach (PERISCOPE) combines a de-stainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This approach provides high-dimensional phenotypic profiles of individual cells, while simultaneously enabling interrogation of subcellular processes. Our atlas reconstructs known pathways and protein-protein interaction networks, identifies culture media-specific responses to gene knockout, and clusters thousands of human genes by phenotypic similarity. Using this atlas, we identify the poorly-characterized disease-associated transmembrane protein TMEM251/LYSET as a Golgi-resident protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes, showing the power of these representations. In sum, our atlas and screening technology represent a rich and accessible resource for connecting genes to cellular functions at scale.