Establishing genetic code expansion as a tool to study neuronal circuit function in an animal

Project Information

UAAWORM

Grant agreement ID: 679990

DOI

10.3030/679990

Project closed

EC signature date 17 March 2016

Start date 1 April 2016

End date 30 September 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 520,00

EU contribution

€ 1 499 520,00

Coordinated by

THE UNIVERSITY OF EDINBURGH
United Kingdom

Periodic Reporting for period 4 - UAAWORM (Establishing genetic code expansion as a tool to study neuronal circuit function in an animal)

Reporting period: 2020-10-01 to 2022-09-30

Summary of the context and overall objectives of the project

The aim of our research is to take an established multicellular model organism with an artificially re-programmed genetic code and develop it into a system to tackle fundamental biological questions. We will initially focus on the nervous system of the nematode worm C. elegans.

We have previously introduced genetic code expansion to C. elegans. It allows the site-specific introduction of chemically synthesized non-canonical amino acids (ncAA) into proteins of interest.

In the past years the field of genetic code expansion has seen significant progress, allowing the incorporation of a growing range of ncAA in diverse systems. While the use of ncAA is still in its infancy, it is beginning to significantly impact the study of previously intractable biological problems. ncAA have been used to site specifically install post-translational modifications, introduce chemical handles that allow the site specific labeling of proteins, and create photo-activateable versions of proteins in vivo using photo-caged amino acids.
Many biological phenomena such as development, ageing or the functioning of nervous systems can only be studied in a multicellular context. We are currently one of very few labs world-wide, establishing the use of ncAA outside single celled systems. We are developing a new type of toolset to study biological processes inside whole multicellular organisms with a level of precision that goes far beyond the current state of the art. Our tools are being applied by us and others to study neurobiological questions, focusing on the contribution of single cells and individual connections to the functioning of neural circuits.

The tools we have developed and are continuing to develop are likely to have significant impact in the life sciences. While we are initially developing these tools in and for C. elegans, they will be transferrable to other systems, since the genetic components we use are functional in all eukaryotic model organisms.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our work has established genetic code expansion as a new technology for the study of biology in multicellular organisms. We have used photo-caged amino acids to precisely control protein activity with light in living organisms. This has allowed us to develop tools to, for example, control gene expression with single-cell precision, using a targeted laser to switch genes on or off.

The tools we have developed are beyond the state of the art we have used them to perform analysis of a neural circuit in freely moving C. elegans at a level not possible before.

Transgenic C. elegans incorporating a non-canonical amino acid into a fluorescent protein