Optical pooled screening (OPS) is a type of high-content single-cell genetic screen that profiles the phenotypes of individual cells by optical microscopy. The phenotypic profile of each cell is linked to one or several genetic features by in situ genotyping. OPS is used to determine the effect of genetic elements on the characteristics of cells and tissues. Single-cell screening methods like OPS have been adopted by the biotechnology industry for applications in drug development. High-content pooled single-cell genetic screens became available as a functional genomics technique starting circa 2016. While the genetic intervention (also known as a "genetic perturbation" in CRISPR screening) can be of any type that can be associated with a genetic sequence in the cell, including modifications in protein-coding or regulatory sequences, CRISPR systems are the most common methodology for affecting genetic perturbations in OPS efforts. The high-content nature of OPS data enables screens for cellular phenotypes not considered prior to data generation and in-depth analysis of the primary screening data to classify and prioritize screening hits. As an intrinsically single-cell-resolved approach, OPS is recognized as capable of identifying perturbation effects on the distribution of single-cell phenotypes across cells. Researchers use OPS to visually assess how gene disruptions and other genetic perturbations cause changes in cellular characteristics like morphology by Cell Painting, protein localization, or intracellular signaling via transduction of signals detected by biochemical receptors in the cell. OPS requires in situ genotyping, for example by in situ sequencing the perturbation in each cell or a nucleotide sequence "barcode" (analogous to the UPC barcode) that links image-based cell phenotypes to specific genetic alterations at the single-cell level. OPS is used in functional genomics, drug discovery, and disease research.
Context OPS is one of two approaches (the other being single-cell next-generation sequencing (NGS)) available to generate high-content single-cell screening data. High-content single-cell functional genomic screens differ from previously established pooled genetic screening approaches relying on enrichment of perturbation identifier frequency in selected versus non-selected or original cell populations. In contrast, high content single-cell screens like OPS match cell phenotypes and perturbation identifiers at the single-cell level, enabling characterization and possible classification of phenotypes post-hoc based on the primary screening data output. Perturbed cell phenotypes are interpreted based on the nature of the perturbations enriched in a phenotypic class, or a quantitative trait can be directly mapped to genetic alteration in a regulatory or coding sequence. In contrast to NGS approaches for high-content single-cell screening OPS directly reads out cellular structures, dynamic molecular/cellular functionality in live cell settings, and can achieve high resolution of cell states. As an imaging method, OPS is applicable where spatial relationships are relevant, for example, the subcellular distribution or localization of organelles or molecular components, and spatial relationships among cells. Imaging assays can also score cell non-autonomous phenotypes such as cell-cell interaction phenotypes, tissue context-dependent phenotypes, and the effect genes have outside the cell. As a live cell imaging method, OPS enables studies of cellular dynamics using advanced imaging modalities, such as single molecule fluorescence microscopy. The capability of OPS to connect the phenotype of each cell in the pooled library to its genotype distinguishes OPS from imaging based pooled enrichment screens such as robotic picking, Visual Cell Sorting, CRISPR-based microRaft followed by guide RNA identification (CRaft-ID), single-cell isolation following time-lapse imaging (SIFT), AI-photoswitchable screening (AI-PS), optical enrichment, image-enabled cell sorting (ICS), and Photopick. These methods all work by segregating cell populations according to pre-specified single-cell image characteristics and bulk readout perturbation identifier abundance in the segregated populations.
History The concept of obtaining image-based phenotypes from pooled cell libraries followed by in situ genotyping of individual cells—now referred to as optical pooled screening (OPS)—was first introduced in 2016,, i.e. concurrently with single-cell screening methods based on NGS, i.e. Perturb-seq, CRISP-seq, and CROP-seq.. The first papers on OPS appeared the year after. One report described a small CRISPR interference screen that perturbed different components regulating a fluorescent reporter protein. In this study, the live-cell phenotyping step was followed by FISH-based readout of barcodes expressed by T7 RNA polymerase from the same plasmid as the CRISPR single guide RNA (sgRNA). Another early report described an OPS with a bacterial library of mutated fluorescent proteins also followed by FISH-based readout of barcodes. Applications in human cells with CRISPR perturbations were subsequently reported with readout of thousands of sgRNA CRISPR perturbations by in situ sequencing of sgRNA and barcode sequences amplified from mRNA using a molecular inversion probe and rolling circle amplification (RCA) and sequencing by synthesis chemistry; and in another example, readout of >100 sgRNA perturbations by FISH. Protein epitopes have also been applied to encode genomic perturbations for enrichment and in vivo OPS with readout from tissue sections. A genome-wide scale loss-of-function CRISPR OPS in human cells was reported in 2023 and included high-content phenotypes recorded from >10 million cells assigned to one of 80,408 sgRNA perturbations. Other genome-wide OPS datasets were reported for infection of human cells by filoviruses, cell signaling, and morphological characterization under different culture conditions. New protocols for nucleotide-level barcode readout incorporate "Zombie" in situ T7 RNA polymerase-driven in vitro transcription for amplification or pre-amplification of OPS readout. A recent application of OPS is genome-wide tracking of chromosome loci over the cell cycle.
Methodology
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