Cell‐Lineage Tracing in Zebrafish Embryos with an Expanded Genetic Code

Abstract

Cell-lineage tracing is used to study embryo development and stem-cell differentiation as well as to document tumor cell heterogeneity. Cre recombinase-mediated cell labeling is the preferred approach; however, its utility is restricted by when and where DNA recombination takes place. We generated a photoactivatable Cre recombinase by replacing a critical residue in its active site with a photocaged lysine derivative through genetic code expansion in zebrafish embryos. This allows high spatiotemporal control of DNA recombination by using 405 nm irradiation. Importantly, no background activity is seen before irradiation, and, after light-triggered removal of the caging group, Cre recombinase activity is restored. We demonstrate the utility of this tool as a cell-lineage tracer through its activation in different regions and at different time points in the early embryo. Direct control of Cre recombinase by light will allow more precise DNA recombination, thereby enabling more nuanced studies of metazoan development and disease.

A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch

“…The tRNA synthetase/tRNA CUA pair allowed the incorporation of the synthetic amino acid in place of the nonsense mutation, and the resulting enzyme was inactive unless it was irradiated with violet or ultraviolet light. This strategy successfully controlled recombination in cultured human cells ( Luo et al, 2016 ) and zebrafish embryos ( Brown et al, 2018 ). We note that it presents several caveats: its combination of chemistry and transgenes is complex to implement, the presence of the tRNA synthetase/tRNA CUA pair can generate off-target artificial C-terminal tails in other proteins by bypassing natural stop codons, and violet/ultraviolet light can be harmful to cells.…”

Optical control of protein phosphatase function

“…In order to abrogate any potential for substrate sequestration by a caged phosphatase, we next explored the possibility of optically controlling the phosphatase:substrate interface. While optical control of protein active sites through site-specific caging group installation has been used to study a wide range of enzymatic functions (e.g., kinase 3–6 , polymerase 30 , nuclease 31 , recombinase 32 , helicase 33 , and others) 34 , optical control of protein-protein interactions has remained underexplored. In this context of transitioning from optical control of enzymatic function to optical control of protein-protein interactions, caged amino acid mutagenesis represents an advantage over other optogenetic approaches, since the position of the caging group is simply defined by the position of the TAG codon and thus can be readily moved to other sites on the protein, e.g., from the active site to the protein surface.…”

A straightforward approach for bioorthogonal labeling of proteins and organelles in live mammalian cells, using a short peptide tag

“…As with other labeling approaches, GCE-tag-based bioorthogonal labeling can be combined with conventional labeling approaches for multicolor labeling and can be tailored for super-resolution microscopy [26]. Moreover, the approach can potentially be expanded to labeling endogenous proteins using genome-editing techniques and can be further applied to recently reported GCE-modified tissues and organisms [27][28][29][30][31]. Additionally, a variety of ncAAs that carries different chemical functionalities has been genetically encoded in mammalian cells [8,[32][33][34][35].…”