Abstract
Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.
Highlights
Extracellular interactions by cell surface receptor proteins direct important biological processes such as tissue organization, host-pathogen recognition, and immune regulation
This approach of identifying interactions mediated by membrane receptors by genetic screens is suitable for researchers that have a focused interest on individual ligands because it avoids the need to compile large libraries of complementary DNAs (cDNAs) or recombinant proteins. This assay consists of three major steps: 1) Highly avid recombinant protein binding probes consisting of the extracellular regions of a receptor of interest are produced and used in fluorescence-based flow cytometry-based binding assays; 2) The binding assays are used to identify a cell line that expresses the interaction partner of the recombinant protein probe; 3) A Cas9-expressing version of the cell line that interacts with the protein of interest is produced and a genome-scale CRISPR/Cas9-based knockout screen is performed (Figure 1)
The binding behavior of RH5 was affected by both heparan sulphate and its known receptor BSG24 (Figure 3C), whereas TNFRSF9 bound to its known receptor TNFSF9 and did not lose binding upon preincubation with soluble heparin
Summary
Extracellular interactions by cell surface receptor proteins direct important biological processes such as tissue organization, host-pathogen recognition, and immune regulation. This approach of identifying interactions mediated by membrane receptors by genetic screens is suitable for researchers that have a focused interest on individual ligands because it avoids the need to compile large libraries of cDNAs or recombinant proteins This assay consists of three major steps: 1) Highly avid recombinant protein binding probes consisting of the extracellular regions of a receptor of interest are produced and used in fluorescence-based flow cytometry-based binding assays; 2) The binding assays are used to identify a cell line that expresses the interaction partner of the recombinant protein probe; 3) A Cas9-expressing version of the cell line that interacts with the protein of interest is produced and a genome-scale CRISPR/Cas9-based knockout screen is performed (Figure 1). General protocols for performing genome-scale screens have already been described[20,21], the protocol mainly focus on the specifics of performing flow cytometry-based recombinant protein binding screens using the CRISPR/Cas[9] knockout screening system using the Human V1 ("Yusa") library[18]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
