Biology subjects

Greiss, S.

Publications and source records attributed to Greiss, S..

5 recordsLinked to original sources

bioRxiv2026

Expanding the Genetic Code of an Animal with Two Non-Canonical Amino Acids

Expanding the genetic code of multicellular organisms for the incorporation of non-canonical amino acids has thus far been limited to a single non-canonical amino acid at a time. To overcome this limitation, we establish the pyrrolysyl-tRNA-synthetase (PylRS)/tRNAPyl pair from Methanomethylophilus alvus in Caenorhabditis elegans and show that it is highly efficient and mutually orthogonal to the Methanosarcina mazei PylRS/tRNAPyl pair previously established in the worm. We furthermore establish a set of mutually orthogonal blank codons for use in C. elegans that allow the simultaneous and independent incorporation of two ncAAs for the first time in an animal. We then engineer the non-canonical amino acid specificity of M. alvus PylRS to incorporate several photocaged ncAAs. Taken together, this allows expression of photocaged variants of FLP and Cre using two different photocaged amino acids, enabling us to optically control the expression of two target genes.

synthetic biology↗

bioRxiv2025

Photocaged FLP Recombinase for Precise Spatio-temporal Control of Gene Expression

The ability to precisely control gene expression is fundamental to studying biological processes. Using site-specific recombinases such as FLP, gene expression can be controlled, albeit with limited spatiotemporal precision. We develop a photocaged FLP recombinase which can be precisely controlled using light, and we demonstrate its efficacy in C. elegans. We use genetic code expansion to incorporate photo-caged amino acids into FLP, replacing critical residues in the active site with their photocaged counterparts. Photocaged FLP displays no detectable background activity, and brief illumination, can be used to activate FLP with near 100% efficiency. We show that photocaged FLP can be activated by light between 365 nm and 435 nm, and that it is not activated by light above 450 nm, making it fully compatible with wavelengths commonly used for imaging and optogenetics. Furthermore, we demonstrate that photocaged FLP can be used to switch on expression of target genes in individual cells within the animal using a standard 405 nm microscope mounted laser to deliver the activating light. Activation by laser requires illumination times of less than 10 ms per cell. Thus, we have developed a straightforward and efficient tool to precisely control gene expression in the multicellular organism C. elegans.

synthetic biology↗

bioRxiv2021

Using a Quadruplet Codon to Expand the Genetic Code of an Animal

Genetic code expansion in multicellular organisms is currently limited to the use of repurposed amber stop codons. Here we introduce a system for the use of quadruplet codons to direct incorporation of non-canonical amino acids in vivo in an animal, the nematode worm Caenorhabditis elegans. We develop hybrid pyrrolysyl tRNA variants to incorporate non-canonical amino acids in response to the quadruplet codon UAGA. We demonstrate the efficiency of the quadruplet decoding system by incorporating photocaged amino acids into two proteins widely used as genetic tools. We use photocaged lysine to express photocaged Cre recombinase for the optical control of gene expression and photocaged cysteine to express photo-activatable caspase for light inducible cell ablation. Our approach will facilitate the routine adoption of quadruplet decoding for genetic code expansion in eukaryotic cells and multicellular organisms.

synthetic biology↗

bioRxiv2021

Generation of photocaged nanobodies for in vivo applications using genetic code expansion and computationally guided protein engineering

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.

synthetic biology↗

bioRxiv2020

Optically splitting symmetric neuron pairs in C. elegans

Synthetic strategies for optically controlling gene expression may enable the precise spatiotemporal control of genes in any combination of cells that cannot be targeted with specific promoters. We develop an efficient genetic code expansion system in C. elegans and use it to create a photo-activatable Cre recombinase. We laser-activate Cre in single neurons within a bilaterally symmetric pair to selectively switch on expression of a loxP controlled optogenetic channel in the targeted neuron. We use the system to dissect, in freely moving animals, the individual contributions of the mechanosensory neurons PLML/PLMR to the C. elegans touch response circuit, revealing distinct and synergistic roles for these neurons. We thus demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs and dissect behavioural outputs of individual neurons that cannot be genetically targeted.

neuroscience↗