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Summary: The interaction between cell surface receptors and their ligands is crucial for intercellular communication. However, current techniques for identifying direct receptor-ligand interactions remain limited. Here, we present a protocol to identify receptors of secreted proteins using a genome-scale CRISPR-Cas9 knockout genetic screening approach. We describe steps for creating a single-guide RNA (sgRNA) lentivirus library, infecting stable Cas9-MCF7 cells, staining with tagged Cholesin, and sorting non-binding cells via flow cytometry. We then detail procedures for extracting DNA, amplifying sgRNAs, and sequencing.For complete details on the use and execution of this protocol, please refer to Hu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
