Curated Optogenetic Publication Database

Abstract: Domain insertion engineering is a powerful approach to juxtapose otherwise separate biological functions, resulting in proteins with new-to-nature activities. A prominent example are switchable protein variants, created by receptor domain insertion into effector proteins. Identifying suitable, allosteric sites for domain insertion, however, typically requires extensive screening and optimization. We present ProDomino, a machine learning pipeline to rationalize domain recombination, trained on a semisynthetic protein sequence dataset derived from naturally occurring intradomain insertion events. ProDomino robustly identifies domain insertion sites in proteins of biotechnological relevance, which we experimentally validated in Escherichia coli and human cells. Finally, we used light- and chemically regulated receptor domains as inserts and demonstrate the rapid, model-guided creation of potent, single-component opto- and chemogenetic protein switches. These include novel CRISPR-Cas9 and -Cas12a variants for inducible genome engineering in human cells. Our work enables one-shot domain insertion engineering and substantially accelerates the design of customized allosteric proteins.

Abstract: Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.