![]() For example, consider a CRISPR-Cas9 gene knockout (CRISPR KO) screen in which a Cas9 nuclease is directed by a sequence-programmable guide RNA (gRNA) to a complementary genomic target, generating mutations that ablate function of the target gene. Today, many screening approaches apply targeted genotypic perturbations across an otherwise constant genetic background, such that differential phenotypes can be directly attributed to the perturbation. FACS, fluorescence-activated cell sorting IF, immunofluorescence scRNA-seq, single-cell RNA sequencing scATAC-seq, single-cell assay for transposase-accessible chromatin using sequencing CyTOF, cytometry by time-of-flight. ![]() (F) Single cell profiling methods for genetic screening include single-cell sequencing approaches, CyTOF using protein barcodes, and microscopy-based phenotyping with in situ genotyping. Individual cells are assigned both perturbations and multidimensional phenotypic measurements. (E) Profiling screens subject a complete cell library to profiling. (D) Cells can be enriched through a fitness advantage, fluorescence-activated cell sorting, or one of several approaches to isolate cells based on microscopy-defined features. Perturbation enrichment is determined by comparing the abundance of perturbation barcodes in the cell library before and after selection using next generation sequencing. ![]() (C) Enrichment screens subject an initial cell library to an enrichment process to select for a phenotype of interest. Arrayed screens can embody either of these phenotype–genotype associations. Pooled enrichment screens project population averages into a unidimensional phenotypic space defined by the enrichment criteria. Pooled profiling screens project individual cells into a multidimensional phenotypic space defined by the profiling method. (B) Screening methodologies capture projections of cell phenotypes. (A) Genetic screens seek to map genotypes to the phenotypes they produce. We discuss how these methodologies, together with emerging technologies for genetic perturbation and microscopy-based multiplexed molecular phenotyping, are powering new approaches to reveal genotype–phenotype relationships. Optical screening now offers the scale needed for systematic characterization and is poised for further scale-up. Microscopy-based cellular profiling provides information complementary to next-generation sequencing (NGS) profiling and has only recently become compatible with large-scale genetic screens. Genetic screens with deep single-cell profiling via image features or gene expression programs have the capacity to show how biological systems work in detail by cataloging many cellular phenotypes with one experimental assay. Microscopy remains the most direct approach to exploring the intricate spatial complexity defining biological systems and the structured dynamic responses of these systems to perturbations. Spatial structure in biology, spanning molecular, organellular, cellular, tissue, and organismal scales, is encoded through a combination of genetic and epigenetic factors in individual cells.
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