![]() This is usually done by high-throughput sequencing. The gRNAs are then counted in the pool of cells retained after the challenge. The gene-edited cells are challenged with a selective pressure such as drug treatment, viral infection or cell proliferation, such that the cells compete with each other based on the fitness effect of the engineered genetic perturbations. ![]() The CRISPR–Cas protein is either stably expressed in the cells or ectopically introduced as a plasmid, virus, mRNA or protein. These gRNAs are usually delivered by lentiviral transduction and are integrated into the DNA of the target cells, making it possible to efficiently determine the induced perturbations based on the gRNA sequence. 1), a CRISPR guide RNA (gRNA) library is introduced in bulk into cells, such that individual cells receive different gRNAs and are perturbed according to the gRNA received by the cell. ![]() They have become a popular and productive tool for biological discovery in a broad range of applications 11, 12. This is illustrated by the many successes of forward genetics in cell lines 1 and in model organisms such as flies 2, 3, worms 4, yeast 5, plants 6 and fish 7, and pioneering work in RNA interference (RNAi) screens 8, 9.ĬRISPR screens exploit the efficiency and flexibility of CRISPR–Cas genome editing 10. Genetic screening provides a powerful approach for identifying genes, pathways and mechanisms involved in a given phenotype or biological process. There is a need in molecular biology and biomedical research for open-ended, hypothesis-generating research, in order to discover previously unknown molecular mechanisms. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development - with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species.
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