Engineering programmable enzymes for proteome editing

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Project Summary/Abstract The development of CRISPR/Cas9 technology for genome editing and reprogramming was one of the most important discoveries in the past decade. The ability to deliver instructions to a specific DNA locus enables systematic interrogation of genetic functions in a broad range of species. In contrast to genomic research, our ability to manipulate the proteome, the functional components of a cell, lags far behind. Natural protein-modifying enzymes, such as proteases and kinases, generally lack the exquisite specificity to perform targeted activities directed to a particular protein. A strategy that enables user-controlled, target-dependent enzyme activation would enable new opportunities for interrogating, modulating, and harnessing endogenous protein functions. In this work, we design autoinhibited enzymes that are activated in response to a highly specific antigen- antibody interaction. These novel enzyme architectures are engineered to have a built-in capability to toggle between a target-unbound, enzyme-inactive conformation and a target-bound, enzyme-active conformation. We present a research plan that outlines the development of a target-activated protease, luciferase, and a kinase, and the subsequent application of the engineered enzymes for controlling protein degradation, constructing biosensors, and re-writing cell signaling pathways. These synthetic enzymes adopt regulatory mechanisms entirely different from the endogenous counterparts, enabling wide possibilities for both basic research and synthetic biological applications. 1