Single-Cell Optical Control of Gene Expression in C. elegans
Davis L, Radman I, Goutou A, Tynan A, Baxter K, Xi Z, O’Shea JM, Chin JW, Greiss S. Precise optical control of gene expression in C elegans using improved genetic code expansion and Cre recombinase. Elife 2021;10:e67075. doi: 10.7554/eLife.67075.
Background: Genetic code expansion enables precise optical control of protein function through incorporation of non-canonical amino acids (ncAAs). In Caenorhabditis elegans, this approach allows for spatiotemporal manipulation of gene expression and neuronal activity. However, limitations in ncAA incorporation efficiency and cell-specific targeting have restricted its application in complex neural circuits.
Hypothesis: This study hypothesized that optimizing the genetic code expansion machinery and combining it with photoactivatable Cre recombinase would enable single-cell optical control of transgene expression in C. elegans, permitting functional dissection of individual neurons within bilateral pairs.
Methods: The authors improved ncAA incorporation by adding a strong nuclear export sequence to the pyrrolysyl-tRNA synthetase (PCKRS) and optimizing its cognate tRNA. They developed a light-activated Cre recombinase (optPC-Cre) and applied this LaserTAC system to induce gene expression in specific neurons. Behavioral responses were recorded using the WormLab Imaging System, and locomotion tracking and quantification were performed with WormLab.
Results: NES-tagged PCKRS variants increased ncAA incorporation efficiency, confirmed by fluorescence and western blot analyses. optPC-Cre achieved nearly complete recombination following light activation. Targeting of optogenetic channels to individual PLM neurons revealed that PLML and PLMR act synergistically in producing robust tail touch responses, with asymmetric contributions to habituation upon repeated stimulation.
Conclusions: The study established LaserTAC as a powerful tool for precise, single-cell genetic manipulation in C. elegans, enabling new insights into neural circuit function and offering broad applicability across tissues and model systems.
