High-performance chemical- and light-inducible recombinases in mammalian cells and mice

Abstract

Site-specific DNA recombinases are important genome engineering tools. Chemical- and light-inducible recombinases, in particular, enable spatiotemporal control of gene expression. However, inducible recombinases are scarce due to the challenge of engineering high performance systems, thus constraining the sophistication of genetic circuits and animal models that can be created. Here we present a library of >20 orthogonal inducible split recombinases that can be activated by small molecules, light and temperature in mammalian cells and mice. Furthermore, we engineer inducible split Cre systems with better performance than existing systems. Using our orthogonal inducible recombinases, we create a genetic switchboard that can independently regulate the expression of 3 different cytokines in the same cell, a tripartite inducible Flp, and a 4-input AND gate. We quantitatively characterize the inducible recombinases for benchmarking their performances, including computation of distinguishability of outputs. This library expands capabilities for multiplexed mammalian gene expression control.

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators

“…In particular, light-induced heterodimerization provides a simple and versatile way to control the activity of split enzymes including Cre recombinase [ 109 ], CRISPR-Cas9 [ 18 ], T7 RNA polymerase [ 48 ] and intracellular antibodies (intrabodies) [ 110 ]. Notably, the VVD-based optogenetic tools have been reported to be temperature-sensitive [ 111 , 112 ] and stimulating cells with colder temperatures can also induce dimerization [ 113 ]. Accordingly, the pre-incubation of cells at the experimental temperature [ 47 ], and keeping the temperature constant during cell imaging, are important considerations for this system.…”

Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering

“…Synthetic split proteins are useful tools for biologists 1 . Often, they are created to serve as sensors for protein-protein interaction 2 , detectors for biomolecules 3,4 , molecular switches 5,6 , and logic gates in synthetic circuits [7][8][9][10] . In other instances, proteins are split to be endowed with temporal-spatial 11 or userdefined controls 12 , or to reduce their sizes for viral delivery 13 .…”

“…Doing so could risk perturbations to the protein structure and function. By either approach, the split site design space is often sparsely sampled by educated guesses with split sites tested through trial-and-error 6,23,24 , which is inefficient. A better solution would be to predict split sites computationally from protein crystal structures.…”

A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits

“…However, this is limited by the dynamical complexity and increased burden on cellular resources caused by increasing the number of biological components [ 3 , 35 ]. Furthermore, there are a finite number of recombinases that have been characterised that can be used reliably as inputs [ 36 ], which limits scalability regardless of the difficulty associated with the number of components.…”