Precise optical control of gene expression in C elegans using improved genetic code expansion and Cre recombinase

Abstract

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 improved 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.

Using a quadruplet codon to expand the genetic code of an animal

“…We aimed to construct a system for quadruplet decoding in C. elegans based on the pyrrolysyl-tRNA synthetase (PylRS)/tRNA(Pyl) pair from Methanosarcina species, which is functional in C. elegans ( 10 , 25 ) and has been used for quadruplet decoding in both bacteria and cultured mammalian cells ( 16 , 18 , 21 ) (Figure 1 ).…”

“…As previously described ( 25 ), worm populations were grown to young adulthood and washed off plates using M9 buffer supplemented with 0.001% Triton-X100 to prevent animals from sticking to pipette tips. Animals were allowed to settle, the supernatant was removed and the worms were resuspended in 4× Bolt LDS sample buffer (Thermo Fisher Scientific) supplemented with 10× Bolt Reducing Agent (Thermo Fisher Scientific).…”

“…Here, we present the first method for quadruplet decoding in a multicellular organism, the nematode worm C. elegans . We develop hybrid tRNA variants by fusing anticodon loops optimized for quadruplet decoding ( 16 , 18 , 23 ) to tRNA scaffolds optimized for interaction with the eukaryotic translational machinery ( 24 , 25 ). We use our system to incorporate two distinct ncAAs, photocaged lysine (PCK) and photocaged cysteine (PCC), and show that we can achieve incorporation efficiencies that come close to those achieved using UAG triplet codons.…”

Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

“…An increasing number of specific and efficient FLP and Cre drivers generated by the C. elegans community are facilitating a variety of precise functional assays to interrogate neural circuits [10,13,24,12], cell proliferation and differentiation [11], gene expression programs [14,51] and chromatin organization [15,16]. In this study, we report four new FLP lines that reliably induce recombination specifically in either distal tip cells, the somatic gonad, coelomocytes or in epithelial Pn.p cells.…”

“…Thanks to community efforts, a growing number of characterized and stable Cre and FLP drivers are available, which facilitates the development and implementation of recombinationbased tools [19, 11,20–23]. Most recently, a light-inducible Cre recombinase has been added to the toolbox [24].…”