Development of Novel Optogenetic Tools By Continuous Directed Evolution of Plant Blue-Light-Dependent Interactions

Arabidopsis thaliana Cryptochrome2 (AtCRY2) is a blue-light receptor that mainly regulates plant photomorphogenesis through blue-light-specific interactions with numerous protein partners. Such blue-light-specific interactions have been exploited in optogenetics to manipulated biological events in a timely and precisely manner. AtCRY2-AtCIB1 photodimerizer is one of the best characterized protein pairs for optical control of protein interactions. However, the application of CRY2-CIB1 was impeded by concomitant homodimerization of AtCRY2, and limited dynamic range. To overcome the limitations, this study focused on the development of a novel pair of blue-light-dependent interacting proteins: AtCRY2-AtBIC1. AtBIC1 (Blue-light Inhibitor of Cryptochromes 1) was recently identified as an endogenous inhibitor of AtCRY2. BIC1 interacts with CRY2 and inhibits homodimerization of CRY2 in the blue light. Additionally, the size of BIC1 is very small (only 15 kDa), which could allow gene fusion with minimal impact. We used a novel continuous directed evolution method developed by David Liu’s lab, PACE (Phage Assisted Continuous Evolution), to evolve AtCRY2 to increase its interaction affinity with BIC1 in the blue light. The PACE method coupled random mutagenesis with selection, enabling dozens of rounds of automatic evolution in a single day with minimal human efforts. Using the method, we successfully isolated mutants of CRY2 with stronger interactions with BIC1. To increase the dynamic range and signal-to-background ratio of CRY2-BIC1 dimerizer, a counter evolution method was proposed to reduce background in darkness. Our study could provide improved tools for optical control of protein interactions.