A Scalable Genome-Editing-Based Approach for Mapping Multiprotein Complexes in Human Cells

Mathieu Dalvai, Jeremy Loehr, Karine Jacquet, Caroline C. Huard, Céline Roques, Pauline Herst, Jacques Côté, Yannick Doyon
2015 Cell Reports  
Graphical Abstract Highlights d Streamlined affinity purification of protein complexes using CRISPR/Cas9 and TALENs d Purification of native holoenzymes regulated under physiological conditions d Blueprint for the systematic and unbiased mapping of the human protein interactome SUMMARY Conventional affinity purification followed by mass spectrometry (AP-MS) analysis is a broadly applicable method used to decipher molecular interaction networks and infer protein function. However, it is
more » ... to perturbations induced by ectopically overexpressed target proteins and does not reflect multilevel physiological regulation in response to diverse stimuli. Here, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. We used CRISPR/Cas9 and TAL effector nucleases (TALENs) to tag endogenous genes and purified several DNA repair and chromatin-modifying holoenzymes to near homogeneity. We uncovered subunits and interactions among wellcharacterized complexes and report the isolation of MCM8/9, highlighting the efficiency and robustness of the approach. These methods improve and simplify both small-and large-scale explorations of protein interactions as well as the study of biochemical activities and structure-function relationships.
doi:10.1016/j.celrep.2015.09.009 pmid:26456817 fatcat:khxrbpnpgvbs3efxy6nabuz2ga