A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86 , ZNF236 , and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena—from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function. [Display omitted] • Perturb-seq maps the transcriptional effects of genetic perturbations at genome scale • Transcriptional signatures allow prediction of function for thousands of genes • Single-cell RNA-seq enables the study of complex composite phenotypes like aneuploidy • Analysis of mitochondrial genome expression reveals diverse, stress-specific regulation Unbiased, genome-scaling profiling of genetic perturbations via single-cell RNA sequencing enables systematic assignment of function to genes and in-depth study of complex cellular phenotypes such as aneuploidy and stress-specific regulation of the mitochondrial genome. [ABSTRACT FROM AUTHOR]