Our project focused on developing and using genetic code expansion technology in a multicellular organism
Genetic code expansion allows the site-specific incorporation, into proteins during translation, of non-canonical "designer" amino acids with properties not found in nature and thereby the modifying of endogenous proteins and installing of new functionalities. Examples are the ability to introduce the ability to control proteins with light, or of site-specific chemical and fluorescent labels, sensors, and post-translational modifications.
In the course of the project we have made breakthroughs that resulted in improving the efficiency of genetic code expansion technology in C. elegans by 50-fold (Davis et al, eLife 2021). This transformational step has opened the door to utilising genetic code expansion to develop ground-breaking molecular tools for tackling biological questions in vivo, which cannot be addressed with existing methods.
We have developed a photocaged Cre recombinase that allows the use of light to switch-on expression of target genes in user-defined cells in the animal. This tool allows precise spatiotemporal control of any gene of interest in single cells or any combination of cells irrespective of promoter availability, thereby filling a long-standing methodological gap. We have applied this versatile tool to control activity of single neurons in freely moving animals and dissect their contributions to a neural circuit (Davis et al, eLife 2021).
Other optogenetic tools we have developed include a light activatable Caspase for optical cell ablation (Xi et al, NAR 2021) and light activatable nanobodies (O'Shea et al, ChemBioChem 2022)
Furthermore, we have demonstrated for the first time, the use of quadruplet codons to expand the genetic code of an animal (Xi et al, NAR 2021). Our advance significantly pushes the state of the art and paves the way for the wider adoption of quadruplet decoding in multicellular organisms. Quadruplets offer wide-ranging advantages compared to previous, triplet decoding systems, and importantly allow the concurrent use of several non-canonical amino acids and thus the further expansion of the non-canonical amino acid based genetic toolbox.
