The Integrated Application Of Adenoviral Delivery Of CRISPR/Cas9 And BioID To Detect Protein-protein Interactions In Vivo

Protein-protein interactions are the molecular bases for the performance of its biological functions. The investigation of protein—protein interaction improves our understanding of the metabolic pathways and physiological processes. To date, several strategies have been commonly used in protein interaction studies, including yeast two-hybrid system, GST pulldown and Co-Immunoprecipitation (Co-IP). However, these above strategies involve high numbers of false positive predictions and may not identify the inherent interactions. Therefore, in the present investigation, we applied a novel strategy to identify inherent protein-protein interaction by combining the adenovirus-mediated CRISPR/Cas9 system and BioID method. Briefly, the adenovirus-mediated CRISPR/Cas9 system is used to specifically cut the target gene, which results in double-strand breaks (DSBs) and triggers homologous repair mechanisms while a homologous repair template is provided by adenovirus. BirA*, the core molecule in BioID, is flanked by 1kb homologous arm, which constructs a fusion gene under homology directed repair, resulting in a fusion protein. BirA* can biotinylate proteins which are adjacent to the target protein and can be further identified by mass spectrometer with high resolution and high sensitivity through affinity enrichment. In our study, Cdh1/Fzr1 (FZR1 fizzy/cell division cycle 20 related 1) was used as a target protein for system validation, since a number of Cdh1 substrates have been identified. By using this integrated strategy, the BirA* knock-in cell line has been established and further validated by PCR and immunofluorescence.