Domain Insertion Profiling Reveals Allosteric Hotspots for Protein Engineering

Constructing allosterically regulated protein switches is critical for many synthetic biology applications. However, the rational engineering of allostery remains a challenging problem. Here we describe a methodology – which we term Domain Insertion Profiling (DIP) – that couples the natural process of domain recombination with functional assays and deep sequencing in order to reveal the underlying potential of allostery within a protein. A common need for allosteric regulation is in the construction of ligand-sensitive fluorescent biosensors. As a proof of concept, we show that DIP analysis rapidly identifies allosteric sites within a ligand binding protein and can be used to isolate green fluorescent protein-based biosensors with high dynamic range. DIP can also be used to assess a protein’s ability to functionally accept genetic fusion. In a related set of experiments, we demonstrate how profiling of the programmable nuclease Cas9 reveals numerous unanticipated hotspots for protein engineering and enables the construction of a Cas9 variant capable of reversible, ligand-dependent genome editing.