Arrayed CRISPR/Cas9 Screening for the Functional Validation of Cancer Genetic Dependencies

CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the pro...

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Bibliographic Details
Published inBio-protocol Vol. 12; no. 24
Main Authors Proietti, Ludovica, Manhart, Gabriele, Heyes, Elizabeth, Troester, Selina, Grebien, Florian
Format Journal Article
LanguageEnglish
Published United States Bio-protocol LLC 20.12.2022
Subjects
Guide RNAs
Flow cytometry
CRISPR/Cas9
Cancer genetic dependencies
Cell fitness
Lentivirus
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Summary:CRISPR/Cas9 screening has revolutionized functional genomics in biomedical research and is a widely used approach for the identification of genetic dependencies in cancer cells. Here, we present an efficient and versatile protocol for the cloning of guide RNAs (gRNA) into lentiviral vectors, the production of lentiviral supernatants, and the transduction of target cells in a 96-well format. To assess the effect of gene knockouts on cellular fitness, we describe a competition-based cell proliferation assay using flow cytometry, enabling the screening of many genes at the same time in a fast and reproducible manner. This readout can be extended to any parameter that is accessible to flow-based measurements, such as protein expression and stability, differentiation, cell death, and others. In summary, this protocol allows to functionally assess the effect of a set of 50-300 gene knockouts on various cellular parameters within eight weeks. Leukemia (2021), DOI: 10.1038/s41375-021-01169-6 Graphical abstract.
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ISSN:2331-8325
2331-8325
DOI:10.21769/BioProtoc.4577