Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled 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 and present a framework to power biological discovery with the resulting genotype-phenotype map. 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 the 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 function and cellular behavior. ### Competing Interest Statement JMR consults for Maze Therapeutics and is a consultant for and equity holder in Waypoint Bio. RAS consults for Maze Therapeutics. KA is a consultant for Syros Pharmaceuticals, is on the SAB of CAMP4 Therapeutics, and received research funding from Novartis not related to this work. GLY, NI, FO, and DL are employees and shareholders of Ultima Genomics. MJ consults for Maze Therapeutics and Gate Bioscience. TMN consults for Maze Therapeutics. JSW declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics and Third Rock Ventures. The Regents of the University of California with RAS, TMN, MJ, and JSW as inventors have filed patent applications related to CRISPRi/a screening and Perturb-seq.