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

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.

(a) Flp was split into two fragments at amino acid S and fused to gibberellin-inducible (GAI/GID1) dimerization domains. Split recombinases were transfected along with reporters that yield green fluorescence protein (GFP) expression upon site-specific recombination (b) Constitutive Flp (black square) or a blank vector (gray square) were transfected to indicate highest or lowest expected GFP expression. Split Flp recombinase components (N-terminal or C-terminal halves) fused to chemical inducible dimerization (CID) domains were transfected in all combinations and drug (colored shapes) or no drug (white shapes) was added to the cell culture medium after two hours. (c) Flp was split into two fragments at amino acid S with no fusion to dimerization domains. Split Flp recombinase components were transfected along with reporters that yield green fluorescence protein (GFP) expression upon site-specific recombination. (d) Constitutive Flp (black square) or a blank vector (gray square) were transfected to indicate highest or lowest expected GFP expression. Split Flp recombinase components (N-terminal or C-terminal halves) domains were transfected in all combinations. M.F.I. indicates mean fluorescence intensity measured with arbitrary units (a.u.). Errors bars represent arithmetic standard error of the mean of three transfected cell cultures. (a and b) Cre was split into two fragments at amino acid S and fused to gibberellin-inducible (GAI/GID1) heterodimerization domains at various termini. Split recombinases were transfected along with reporters that yield green fluorescence protein (GFP) expression upon site-specific recombination (c). Constitutive Cre (black square) or a blank vector (gray square) were transfected to indicate highest or lowest expected GFP expression. Split recombinases were also transfected and drug (blue squares) or no drug (white squares) was added to the cell culture medium after two hours. M. Single-cell data represented as smoothed scatterplots are plotted for the 21 hour (a), 52 hour (b) and 100 hour (c) time points captured over the 100 hour time-course of the experiment in Figure  2b. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ). Single-cell data represented as smoothed scatterplots are plotted of the experiment in Figure 2c and 2d. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ).

Supplementary Figure 11. Single-cell fluorescence representation of selected Flp split systems.
Single-cell data represented as smoothed scatterplots are plotted of the experiment in Figure 2e and 2f. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ).
Single-cell data represented as smoothed scatterplots are plotted of the experiment in Figure 2g and 2h. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ).

Supplementary Figure 13. Single-cell fluorescence representation of selected ɸC31 split systems.
Single-cell data represented as smoothed scatterplots are plotted of the experiment in Figure 2i and 2j. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ).

Supplementary Figure 14. Single-cell fluorescence representation of selected TP901 and Bxb1 split systems.
Single-cell data represented as smoothed scatterplots are plotted of the experiment in Figure 2k through 2n. Axes represent the expression of transfection marker, BFP, in molecules of equivalent fluorescein (MEFL) and GFP output, in MEFL, from recombinase reporters. Density of cytometric events are represented ranging from no events (dark blue) to highest density in dark red. White dots represent single event outliers. Events under a BFP MEFL cutoff of 1x10 6 (representing nontransfected or poorly transfected cells) were removed from analysis and a red line at GFP MEFL = 1x10 6 indicates a boundary between cells that are off (below 1x10 6 ) and cells that are on (above 1x10 6 ).