Modular construction of mammalian gene circuits using TALE transcriptional repressors

Abstract

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator-like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.

Figure 1. Design and construction of TALE repressors for mammalian cells

(a) Schematic representation of TALE repressors with repeat di-variable residue (RVD) domains. The repeat domains are shown in orange blocks with RVDs in magenta and the binding specificity of RVDs is shown below. The last domain is a 15 amino acid half repeat domain depicted in a grey box. The green block represents the requirement of a leading repeat that binds T. The blue block represents a nucleus localization signal (NLS). (b) Design of a fluorescent reporter assay for measuring TALER repression fold and orthogonality. Tx: binding site for TALER x. Blue arrow represents a minimal CMV promoter. Lines with arrows indicate up regulation and lines with bars indicate down regulation. (c) Functional assay of TALERs in HEK293 cells. Selected RVD domains and binding sequences are shown on the left. Blue bars represent repression fold and red boxes represent repression percentage. Each bar/box shows mean ± SD from three independent flow cytometry experiments. (d) TALER orthogonality matrix. Each box in matrix represents fluorescent reporter expression measured in co-transfection of indicated TALER protein and promoter.

Figure 2. Design and construction of TALER cascade

(a) Schematic representation of a circuit for measuring transfer curves. pTxBS2 and pTxBS4 are TALER promoters with two binding sites or four binding sites, respectively. (b) Circuit outputs for characterizing transfer curves indicated in (a) at varying Dox concentrations. Each data point shows mean ± SD from three independent replicates. (c) Schematic representation of a cascade circuit. For simplicity, pTxBS2 and pTxBS4 are depicted in the same diagram with two downstream binding sites in pTxBS4 promoter colored with reduced intensity. (d) Measured output from cascade circuit indicated in (c) at varying Dox concentrations. Each cascade is named by concatenating the name of the first TALER, ‘-‘, and the name of the second TALER. Each data point shows mean ± SD from three independent replicates.

Figure 3. Design and construction of TALER sensory switches

(a) Schematics of TALER switching circuits. Each TALER level is monitored by 2A-linked mKate2 or EYFP fluorescence. shRNAs are represented by wiggly lines, and shRNA targets are shown as blocks in brown or grey color. (b) Indicated TALER switches were co-transfected into HEK293 cells. Representative flow cytometry scatter plots measured 48 h post-transfection are shown in a matrix layout, where each row shows the same mKate2-linked TALER used in transfection experiments and each column represents the same EYFP-linked TALER used in transfection experiments. Correlation of the ratio of EYFP to mKate2 is shown in the top-right graph. Each data point shows mean ± SD from three independent replicates. (c) and (d) Setting the states of TALER21-TALER14 switches (c) or TALER21-TALER9 switches (d) by shRNAs. The upper panels represent schematics of TALER switches. Each bar shows mean ± SD of EYFP or mKate2 from three independent replicates. The lower panel represents representative flow cytometry scatter plots measured 48 h post-transfection.

Figure 4. Response of TALER sensory switches to shRNA inputs

(a) Schematic representation of a closed-loop TALER switch with mutual inhibition and the open-loop counterpart without mutual inhibition. (b) TALER switch behavior in response to varying amounts of shRNA inputs. Ratio of TALER9 to TALER14 (or TALER10) used in transfection experiments is indicated as 1:1 or 2:1. Each data point shows mean ± SD of fluorescent reporter from three independent replicates. Closed: closed-loop circuit; Open: open-loop circuit.

Figure 5. Connecting microRNAs to regulate TALER sensory switches enables cell-type classification

(a) Experimental outline. HEK293 cells (purple circle) that express a synthetic shRNA-FF4 and iRFP along with HeLa cells (blue circle) that express TagBFP were transfected with TALER9-TALER14 switches in response to indicated microRNAs or shRNA-FF4. (b) TALER switches controlled by HeLa-specific miR21 and indicated HEK293-specific microRNA. The bar chart shows the fraction of engineered HEK293 and HeLa in EYFP⁺ or mKate2⁺ cell population. Each bar shows mean ± SD from three independent replicates. FACS data is shown in the scatter plot. The upper row shows distribution of EYFP⁺ cells on iRFP-TagBFP scatter plots, and the lower row shows distribution of mKate2⁺ cells on iRFP-TagBFP scatter plots. Circles indicate engineered HEK293 or HeLa cell population.