Using De Novo Designed Protein Switches in C. elegans

Abstract

The ‘Latching Orthogonal Cage/Key pRoteins’ (LOCKR) system is a de novo designed protein Switch and Key. The Switch functions to cage a functional protein sequence, such as a degron, in the off or “locked” state. Upon Key addition, the Switch is turned on or “unlocked,” which exposes the degron, simultaneously degrading the Switch and any proteins fused to the Switch via the proteasome. Key expression can be tailored for tissue‐specific expression, which gives LOCKR exquisite spatial protein control. LOCKR provides novel post‐translational control over a protein of interest but has not yet been applied to a multicellular organism. We are developing the LOCKR technology for conditional protein depletion in the model organism C. elegans. Although genetic RNAi and similar gene editing techniques benefit from ease of delivery and can target essentially any gene, they have various limitations, including 1) slow protein turnover can prohibit use of RNAi; 2) genetic perturbation of essential genes via genetic deletion prevents characterization of later functions. The degronLOCKR system requires two parts: 1) a gene fused to the ‘degronSwitch’ and 2) an inducer peptide (the Key). The Switch can be fused to any gene of interest using CRISPR‐Cas9 gene editing. We fused degronSwitch to dhc‐1 (dynein heavy chain), an essential cytoplasmic motor protein along with mScarlet and a 3xFLAG tag to aid in protein quantitation via microscopic and Western analysis. We also cloned a negative control, which lacks degronSwitch, to analyze the effect of inserting the Switch in vivo. To complement the degronSwitch, we designed six tissue‐specific Keys for Mos‐Single Copy Insertion (MosSCI), which allows for spatial control of degronSwitch protein degradation. We predict that ubiquitous expression of Key with the degronSwitch fused to dhc‐1 will result in embryonic lethality, whereas germline‐specific Key will permit viability but result in meiotic defects. These phenotypes are easily identifiable and serve as effective proof of concept. Future work includes expanding the LOCKR system to incorporate temporal control of the degronSwitch fusion protein by toggling the Key on and off using heat‐shock promoters and RNAi against the Key. This work establishes a novel and tissue‐specific method for regulating protein function in an animal model.