Optimizing the cODC degron for Protein Control in Nematode Worms

Abstract

Caenorhabditis elegans, a nematode worm, is a model multicellular organism for the study of development and disease. Current methods of controlling essential and pleiotropic gene product levels in C. elegans suffer from limitations, including incomplete penetrance, false negatives, and the inability to differentially control multiple genes with the same system simultaneously. Genetic control methods such as loss‐of‐function alleles and CRISPR‐Cas9 targeted genes have proved successful at deducing the relationship between gene function and phenotype; but they lack the ability to investigate genes essential for viability, fertility, and pleiotropic genes that have dissimilar roles in different tissues during the nematode’s lifespan. RNAi is an effective method for studying gene function in C. elegans, it is easy to deliver, can target virtually any sequence, and can be administered at any time during development. However, RNAi suffers from several limitations including: a significant portion of genes cannot be adequately silenced, RNAi cannot enter equally into all tissues, there is a risk of off‐target effects, and the observation that RNAi phenotypes may be delayed if existing proteins persist for long periods of time. Protein degradation methods can overcome many limitations of the genetic methods by genetic fusion of the gene of interest to an inducible protein degradation domain. Some protein control methods lack the ability to target control of protein degradation to specific tissues, while all current methods are limited by the use of a single input signal to control degradation. The overarching goal of this study is to develop a system for complex control of protein function in nematodes using the Latching Orthogonal Cage/Key pRoteins (LOCKR) system, a de novo‐designed protein Switch comprised of a Cage and a Latch that is capable of caging any linear protein sequence. To conditionally control protein levels in nematodes, we will fuse target genes to LOCKR with a cODC degron sequence encoded in the Latch. The interaction of the Cage with the Latch blocks the accessibility of the degron. Introducing a peptide Key releases the Latch, resulting in exposure of the degron sequence and degradation of fused proteins. Here we describe the development of a bacterially‐expressed LOCKR system suitable for in vitro degron optimization. The mEGFP‐degronLOCKR fusion expresses in bacteria and can be purified using immobilized metal affinity chromatography. We hypothesize that when Switch is activated by the Key and the degron is exposed, the mEGFP‐degronLOCKR construct will be degraded in C. elegans lysate. We further predict that various mutations in the degron will affect the rate of target protein degradation. This study will allow more detailed and complex studies of development and disease in C. elegans and may be applicable to other multicellular organisms.