Light-activateable apoptosis via genetic code expansion as an in-vivo single-cell ablation tool in Caenorhabditis elegans

Abstract

Natural proteins are biopolymers built from a limited variety of canonical amino acids that are encoded by corresponding triplet codons. Genetic code expansion via amber suppression enables me to install (incorporate) in a target protein a “designer” amino acid beyond canonical amino acids, at the site of a pre-introduced amber stop codon. This is through expression in the host cell of an orthogonal pair, consisting of an aminoacyl-tRNA synthetase and an amber-suppressing tRNA (tRNACUA) evolved for the non-canonical amino acids (NCAA). Site-specific incorporation of NCAA endows target proteins with new properties, enabling protein measurement and/or manipulation in ways that are otherwise impossible.

Photo-caged cysteine (PCCys) as a useful NCAA has not been used in any animal before. By using a PCCRS/tRNAPylCUA pair evolved from a PylRS/tRNAPylCUA pair, I introduced (PCCys) into protein synthesis of multicellular model species Caenorhabditis elegans (C. elegans). I demonstrated this incorporation of PCCys by expressing a fluorescent reporter either throughout the nematode or in two different neuronal classes.

I used site-specific PCCys incorporation to develop a light-activatable caspase for precisely ablating cells (especially neurons) in living worms. Cell ablation has been widely adopted in studies on C. elegans cell lineage and cell functions. Common ablation methods include high-powered laser ablation, genetic ablation and optogenetic ablation. However, they are unable to ablate single cells in fully developed worms. Caspase is a core executor of apoptosis of both C. elegans and human cells. I designed and engineered a photo-caged caspase from human Caspase-3 by replacing its catalytic cysteine with PCCys. 365-nm UVA illumination removes the caging group of PCCys in the caged caspase, thereby activating the caspase to induce apoptosis of the cell(s) targeted. I succeeded in using global UV illumination to activate respective apoptosis events of oxygen-sensing neurons, touch receptor neurons and muscular cells in adult worms. Also, I demonstrate that individual adult neurons can be selectively targeted and efficiently killed with the use of a microscope-mounted 365-nm laser. With this better spatiotemporal control than other ablation methods, our approach is likely to facilitate future C. elegans studies with unprecedented specificity and precision.