CrY2H-seq: a massively-multiplexed assay for deep coverage interactome mapping

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively-multiplexed yeast two-hybrid method, CrY2H-seq, that uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins, and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated combinatorial interactions among plant transcription factors. By performing ten independent CrY2H-seq screens each testing 3.6 million interaction combinations, and reporting a deep coverage network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq’s improved capacity, efficiency, and sensitivity over existing technologies. In addition to recapitulating one third of previously reported interactions derived from diverse methods, we expand the number of reported plant transcription factor interactions by three-fold, revealing previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.