Biology subjects

Tous, C.

Publications and source records attributed to Tous, C..

3 recordsLinked to original sources

bioRxiv2025

Multiplexing Light-Inducible Recombinases to Control Cell Fate, Boolean Logic, and Cell Patterning in Mammalian Cells

Light-inducible regulatory proteins are powerful tools to interrogate fundamental mechanisms driving cellular behavior. In particular, genetically encoded photosensory domains fused to split proteins can tightly modulate protein activity and gene expression. While light-inducible split protein systems have performed well individually, few multichromatic and orthogonal gene regulation systems exist in mammalian cells. The design space for multichromatic circuits is limited by the small number of orthogonally addressable optogenetic switches and the types of effectors that can be actuated by them. We developed a library of red light-inducible recombinases and directed patterned myogenesis in a mesenchymal fibroblast-like cell line. To address the limited number of light-inducible domains (LIDs) responding to unique excitation spectra, we multiplexed light-inducible recombinases with our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform. Multiplexed optogenetic tools will be transformative for understanding the role of multiple interacting genes and their spatial context in endogenous signaling networks.

molecular biology↗

bioRxiv2024

Complex optogenetic spatial patterning with split recombinase

Light is a powerful and flexible input into engineered biological systems and is particularly well-suited for spatially controlling genetic circuits. While many light-responsive molecular effectors have been developed, there remains a gap in the feasibility of using them to spatially define cell fate. We addressed this problem by employing recombinase as a sensitive light-switchable circuit element which can permanently program cell fate in response to transient illumination. We show that by combining recombinase switches with hardware for precise spatial illumination, large scale heterogeneous populations of cells can be generated in situ with high resolution. We envision that this approach will enable new types of multicellular synthetic circuit engineering where the role of initial cell patterning can be directly studied with both high throughput and tight control.

synthetic biology↗

bioRxiv2023

Orthogonal inducible control of Cas13 circuits enables programmable RNA regulation in mammalian cells

RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13s that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineered Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13s, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.

synthetic biology↗