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.

Fig. 1

Screens of split locations yield small-molecule-dependent recombination responses. Cre, Flp, VCre, ɸC31, and TP901 recombinases (a–f) were split into two fragments at amino acid S and fused to gibberellin-inducible (GAI/GID1) or rapalog-inducible (FKBP/FRB) heterodimerization domains. Splits were made at various amino acid locations; pink, blue, and gray shaded regions indicate α-helices, β-sheets, or undetermined structures, respectively. Split recombinases were transfected along with reporters that yield green fluorescence protein (GFP) expression upon site-specific recombination (g–l). Constitutive recombinases (black square) or a blank vector (gray squares) were transfected to indicate highest or lowest expected GFP expression. Split recombinases were also transfected, and drug was either added (green squares) or not added (white squares) to the cell culture medium after 2 h. MFI indicates mean fluorescence intensity measured with arbitrary units (a.u.). Errors bars represent arithmetic standard error of the mean of three transfected cell cultures. Source data are provided as a Source Data file

Fig. 2

Swapping chemical-inducible dimerization domains yields a large repertoire of small-molecule inducible recombinases. a A signal-to-noise (SNR) metric can be used to capture distinguishability of on and off states, accounting for both the absolute difference in mean signal expression and spread (noise) of the distributions. SNR dynamics in units of decibels (dB) were captured over 100 h (b) post transfection for particular amino acid splits of inducible Cre, Flp, and ɸC31 systems incorporating the gibberellin (GIB), rapalog (RAP), and abscisic acid (ABA) dimerization domains GAI/GID1, FKBP/FRB, and PYL/ABI, respectively. A 4-hydroxytamoxifen (4OHT)-inducible pCAG-ER^T2-Cre-ER^T2 construct is also included in b. Various splits of Cre (c, d), Flp (e, f), VCre (g, h), ɸC31 (i, j), TP901 (k, l), and Bxb1 (m, n) were incorporated with the GIB-, RAP-, and ABA-associated dimerization domains. Measurements of GFP mean fluorescence intensity (MFI) in units of molecules of equivalent fluorescein (MEFL) and SNR were made for plus drug (colored blue, purple, and pink squares corresponding to gibberellin, rapalog, and abscisic acid added 2 h post transfection) and minus drug (white squares) conditions. Constitutive recombinases (black squares) and blank vectors (gray squares) were also transfected as a comparison. Red diamonds indicate SNR score. Error bars of MFI indicate the geometric standard deviation of means for three transfected cell cultures. Source data are provided as a Source Data file

Fig. 3

Light- and temperature-inducible recombinases. a A schematic showing a split Cre fused to nMag and pMag blue light-inducible dimerization domains. b Pulses indicating the changing light (left) and temperature (center and right) conditions. Plots indicate GFP mean fluorescence intensity (MFI) of various nMag/pMag-tagged split Cre (c) and Flp (d) recombinases under blue light (blue squares), room temperature (pink squares), and refrigeration (purple squares). Black squares indicate samples treated with constant temperature and dark conditions. PA-Cre indicates expression of a previously developed blue light-inducible Cre recombinase. Inducible recombinases were compared with samples transfected with constitutive recombinases or a blank vector. Error bars of MFI indicate the arithmetic standard error of the mean between three transfected cell cultures. Source data are provided as a Source Data file

Fig. 4

Protein and DNA-based recombinase logic gates. a A two-input protein-based AND gate is created by splitting Flp recombinase at two locations (S₁ = 27, S₂ = 396) and fusing to the gibberellin (GIB)- and abscisic acid (ABA)-associated chemical inducible dimerization domains. b Plotted results indicate GFP mean fluorescence intensity (MFI) of four conditions of GIB and ABA. c Four chemical-inducible recombinases can be used together to generate a DNA-based 4-input logic gate; these include a gibberellin-inducible split Bxb1 (S = 468), 4-hydroxytamoxifen (4OHT)-inducible ER^T2-Cre-ER^T2, abscisic acid (ABA)-inducible split Flp (S = 396), and rapalog (RAP)-inducible split ɸC31 (S = 571). Drugs were added 2 h post transfection. d Plotted results indicate GFP. MFI of 16 conditions of GIB, 4OHT, ABA, and RAP. Error bars of MFI indicate the arithmetic standard error of the mean between three transfected cell cultures. Source data are provided as a Source Data file

Fig. 5

Applications of split recombinases. a A genetic switchboard using three inducible split recombinases to individually control expression of three therapeutically relevant human cytokines, interferon-γ (IFNγ), interleukin-2 (IL-2), and interleukin-10 (IL-10). Drugs were added 1 day post transfection. b Measurement of supernatant cytokine concentration using enzyme-linked immunosorbent assays (n = 3). c A xenograft mouse model of split recombinases used in a that were transfected in human embryonic kidney (HEK) cells alongside a corresponding luciferase reporter that expresses upon site-specific recombination. Transfected cells are transplanted subcutaneously (s.c.) and drugs or vehicle-only control are injected intraperitoneally (i.p.). d Time-course depicting time of transfection, injections, and imaging. e Images and averaged values of emitted luminescence from three split-recombinase systems. Error bars of mean cytokine and luminescence values indicate the arithmetic standard error of the mean. Source data are provided as a Source Data file