Dial: programmable promoter editing to generate defined, heritable setpoints of gene expression

Abstract

An engineered nucleic acid for regulating levels of gene expression, including in cells is provided. The nucleic acid has a regulatory region with a transcription factor binding site upstream of a transcription start site and separated by a spacer and includes at least one of multiple distinct transcription factor binding sites, a recombination regulation system and / or nested excisable spacers and orthogonal recombination sites. Methods of regulating gene expression are also provided.

Description

[0001] DIAL: PROGRAMMABLE PROMOTER EDITING TO GENERATE DEFINED, HERITABLE SETPOINTS OF GENE EXPRESSION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 651,358 filed May 23, 2024, entitled “DIAL: PROGRAMMABLE PROMOTER EDITING TO GENERATE DEFINED, HERITABLE SETPOINTS OF GENE EXPRESSION,” and U.S. Provisional Application No. 63 / 770,820 filed March 12, 2025, entitled “DIAL: PROGRAMMABLE PROMOTER EDITING TO GENERATE DEFINED, HERITABLE SETPOINTS OF GENE EXPRESSION,” the contents of each of which are hereby incorporated by reference herein in their entirety for all purposes.

[0004] FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under W91 INF- 19-2-0026 awarded by the U.S. Army Research Office, and GM143033 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Synthetic transcription factors induce expression of transgenes by recruiting transcriptional machinery to transcription start sites within synthetic promoters. (Khalil, A. S. et al. A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions. Cell 150, 647-658 (2012); Donahue, P. S. et al. The COMET toolkit for composing customizable genetic programs in mammalian cells. Nature Communications 11, 1-19 (2020); Wu, M.-R. et al. A high-throughput screening and. computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS). Nature Communications 10, 2880 (2019).) The number and affinity of transcription factor binding sites and their distance to the core promoter and transcription start site influence the levels of transgene expression from these synthetic promoter systems (Donahue, P. S. et al. (2020), id). Within a limited range, increasing levels of synthetic transcription factors increases the mean level of expression across a population of cells (Duran, A. G. et al. Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems. ACS Synth. Biol. 11, 2623-2635 (2022).). However, the strong cooperative behavior from these synthetic promoters results in bimodal expression where individual cells exist in either “ON” or “OFF” states (or modes) (as depicted in the schematic of FIG. 1, left panel). Consequently, these synthetic promoters do not allow for the exploration of intermediate levels of expression.

[0008] SUMMARY

[0009] Subtle changes in gene expression direct cells to distinct cellular states. While titrating expression could reveal dose-dependent state regulators, few tools generate unimodal distributions of varying means particularly from a single genetic construct. For instance, titrating activators of inducible promoters often generates bimodal transgene expression without substantially influencing either population mode. An editable promoter system that allows for fine-scale, stable changes in transgene expression has been developed and is disclosed herein. By harnessing a recombinase system, the distance between the binding sites of a transactivator and transcription start site (i.e., binding sites of a synthetic zinc-finger transcription factor and the core promoter) can be fine-tuned. Tessellated binding sites can also be used to allow for multiple synthetic transcription factors to modularly alter the level of expression further. It is demonstrated herein that this system, referred to herein as “DIAL” for its ability to stably “dial” setpoints, generates a tunable range of unimodal setpoints from a single, genetically uniform reporter state. Layering orthogonal recombinase recognition sites with spacers of different lengths expands the number of setpoints within a single construct. It is also shown herein that the setpoints are robust against changes in the levels of the synthetic transcription factors above a threshold. DIAL, in some embodiments, allows for temporally-defined, user-guided control of transgene expression via control of recombinase and transactivators via small-molecules and modRNA. As shown in the Examples, DIAL can be delivered with viral vectors to effectively control expression levels in primary cells. Therefore, DIAL provides a new method to tailor transgene expression levels to probe the influence on cell trajectories and phenotypes, which will be beneficial for gene and cell-based therapies.

[0010] In some aspects, an engineered nucleic acid is provided. In some aspects, the engineered nucleic acid includes a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises at least one of: a) at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator, b) a recombination regulation system for excising at least a portion of the spacer, or c) where the spacer comprises nested excisable spacers and orthogonal recombination sites.

[0011] In some embodiments the regulatory region comprises a) and b). In some embodiments the regulatory region comprises a) and c). In some embodiments the regulatory region comprises b) and c). In some embodiments the regulatory region comprises a), b) and c).

[0012] In some aspects, the engineered nucleic acid includes a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or wherein the regulatory region is upstream of a site for gene insertion, and wherein the regulatory region comprises at least one of: a) at least one type of distinct transcription factor binding sites or multiple tessellated distinct transcription factor binding sites, each specific for a different transactivator, b) a recombination regulation system for excising at least a portion of the spacer, or c) where the spacer comprises nested excisable spacers and orthogonal recombination sites.

[0013] In some embodiments the regulatory region comprises a) and b). In some embodiments the regulatory region comprises a) and c). In some embodiments the regulatory region comprises b) and c). In some embodiments the regulatory region comprises a), b) and c).

[0014] In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises a recombination regulation system for excising at least a portion of the spacer.

[0015] In some embodiments, the excisable portion of the spacer is at least 25 nucleotides in length. In some embodiments, the excisable portion of the spacer is 20- 900, 25-650, 25-500, 50- 900, 50-650, 100-900, 100-650, 200-900, or 200-650 nucleotides in length. In some embodiments, the spacer comprises nested excisable spacers and orthogonal recombination sites. In some embodiments the spacer comprises 2-10 nested excisable spacers and 2-10 orthogonal recombination sites.

[0016] In some embodiments, the recombination regulation system is a tyrosine recombinase or serine recombinase recombination system. In some embodiments, the recombination system comprises at least two recombination sites in the spacer.

[0017] In some embodiments, the recombination regulation system is a Cre-loxP recombination system. In some embodiments, the Cre-loxP recombination system comprises at least two loxP sites in the spacer.

[0018] In some embodiments, the recombination regulation system is a VCre-VloxP recombination system. In some embodiments, the Vcre-VloxP recombination system comprises one or more VloxP sites in the spacer. In some embodiments, the Vcre-VloxP recombination system comprises at least two VloxP sites in the spacer.

[0019] In some embodiments, the recombination regulation system is a Flp-F14 recombination system. In some embodiments, the Flp-F14 recombination system comprises one or more F14 sites in the spacer. In some embodiments, the Flp-F14 recombination system comprises at least two F14 sites in the spacer.

[0020] In some embodiments, the recombination regulation system is a Flp-FRT recombination system. In some embodiments, the Flp-FRT recombination regulation system comprises one or more FRT sites in the spacer.

[0021] In some embodiments, the recombination regulation system is a Bxbl-attB / attP recombination system. In some embodiments, the Bxbl-attB / attP recombination regulation system comprises one or more Bxbl-attB sites in the spacer. In some embodiments, the Bxbl- attB / attP recombination regulation system comprises one or more Bxbl attP sites in the spacer. In some embodiments, the Bxbl-attB / attP recombination regulation system comprises one or more Bxbl-attB sites and one or more Bxbl-attP sites in the spacer.

[0022] In some embodiments, the recombination regulation system is a PhiC31-attB / attP recombination system. In some embodiments, the PhiC31-attB / attP recombination regulation system comprises one or more PhiC31 attB sites in the spacer. In some embodiments, the PhiC31-attB / attP recombination regulation system comprises one or more PhiC31-attP sites in the spacer. In some embodiments, the PhiC31-attB / attP recombination regulation system comprises one or more PhiC31-attB sites and one or more PhiC31-attP sites in the spacer. In some embodiments, the regulatory region further comprises at least one transcription factor binding site. In some embodiments, the regulatory region further comprises multiple transcription factor binding sites. In some embodiments, the multiple transcription factor binding sites are distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, the multiple transcription factor binding sites are tessellated. In some embodiments, the multiple transcription factor binding sites comprise multiple tessellated distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, the regulatory region further comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator.

[0023] In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site(s) upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the spacer comprises nested excisable spacers and orthogonal recombination sites. In some embodiments, each of the nested excisable spacers is at least 100 nucleotides in length. In some embodiments, the spacer comprises 2-10 nested excisable spacers and 2-10 orthogonal recombination sites. In some embodiments, the regulatory region further comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator.

[0024] In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises at least one type of transcription factor binding site.

[0025] In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises multiple transcription factor binding sites. In some embodiments, the regulatory region comprises multiple tessellated transcription factor binding sites. In some embodiments, the regulatory region comprises multiple distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, the regulatory region comprises multiple, tessellated distinct transcription factor binding sites, each specific for a different transactivator. In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises at least one type of transcription factor binding sites, or multiple tessellated distinct transcription factor binding sites, each specific for a different transactivator.

[0026] In some aspects, an engineered nucleic acid provided herein comprises a regulatory region comprised of a transcription factor binding site upstream of a transcription start site and separated by a spacer, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator,

[0027] In some embodiments, the transactivator is a synthetic zinc finger transcription factor. In some embodiments, the transactivator is a small molecule-inducible transactivator. In some embodiments the transactivator is a Tetracycline (TET) activated transactivator. In some embodiments, the transactivator is a doxycycline (DOX) activated transactivator.

[0028] In some aspects, a method of regulating gene expression is provided. The method involves contacting a cell with an engineered nucleic acid as disclosed herein, wherein the engineered nucleic acid comprises a gene downstream of the regulatory region, and contacting the cell with a transactivator and / or a recombinase to regulate expression of the gene. In some embodiments, the recombinase is a Cre recombinase, a VCre recombinase, a Flp recombinase, a BxB 1 recombinase, a PhiC31 recombinase, or a combination thereof. In some embodiments the transactivator is a vector encoding a transactivator protein. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the vector encoding a transactivator protein comprises a constitutive promoter. In some embodiments the vector encoding a transactivator protein comprises an inducible promoter. In some embodiments, the vector does not comprise a promoter. In some embodiments, the vector is a viral vector and does comprise a promoter.

[0029] In some embodiments the transactivator protein is a synthetic zinc finger transcription factor. In some embodiments the transactivator protein is a small-molecule inducible rtTA. In some embodiments, the level of transactivator delivered to the cell is greater than a threshold level to achieve bimodal gene expression. In some embodiments the method further comprises contacting the cell with a transcription factor inhibitor. In some embodiments the transcription factor inhibitor is a zinc finger inhibitor.

[0030] In some embodiments the cell is a primary cell.

[0031] In some embodiments the cell is in a subject.

[0032] In some embodiments the engineered nucleic acid is incorporated in a lentiviral vector.

[0033] In some aspects, a viral vector comprising an engineered nucleic acid as disclosed herein is provided.

[0034] In some aspects, a method of increasing expression of a gene in a cell is provided. In some aspects, the method comprising excising at least a portion of a spacer in a regulatory region of a nucleic acid of the cell, wherein the regulatory region comprises a transcription factor binding site and a transcription start site separated by the spacer, wherein the regulatory region is upstream of the gene, and wherein excising the portion of the spacer increases expression of the gene.

[0035] In some embodiments, excising the portion of the spacer comprises contacting the cell with a recombinase cognate to recombination sites flanking the portion of the spacer. In some embodiments, the regulatory region further comprises at least one transcription factor binding site. In some embodiments, the regulatory region further comprises multiple transcription factor binding sites. In some embodiments, the multiple transcription factor binding sites are tessellated. In some embodiments, the multiple transcription factor binding sites comprise multiple distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, the multiple transcription factor binding sites comprise multiple tessellated distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, the regulatory region further comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator.

[0036] In some embodiments, the method further comprises contacting the cell with a transactivator protein.

[0037] BRIEF DESCRIPTION OF DRAWINGS

[0038] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. For purposes of clarity, not every component may be labeled in every drawing. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure. In the drawings:

[0039] FIG. 1 is a schematic showing the cooperativity of synthetic promoters. In the left panel the synthetic promoters generate bimodal distributions of expression with cells being in either the “ON” or “OFF” state. By generating discrete molecular states as disclosed herein, promoter editing generates stable unimodal expression across a range of modes, as shown in the right panel.

[0040] FIGs. 2A-2F show editable ZF-inducible promoter mediates logic gate for expression. FIG. 2A is a schematic of single cassette delivery generating diversity of states from TF and recombinase combinations. FIG. 2B is a schematic of an editable promoter in which Cre- mediated excision of a floxed 203 bp spacer between the ZF binding sites and YB_TATA minimal promoter will increase expression. FIGs. 2C-2I show data from HEK293Ts transiently transfected with the 203bp-spacer promoter with various target genes. FIG. 2C is a Logic table for how input ZFal and Cre combinations lead to output expected reporter states and experimentally different expression levels shown in histograms sampled from bioreplicates (n=3), gated by a cotransfected marker. FIGs. 2D and 2E show the geometric mean fluorescence intensity (MFI) of a single target gene or multiple target genes, respectively, in combination with ZFal, ZFa2, and Cre. Circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 2F is a schematic showing the PCR procedure to detect editing of the promoter.

[0041] FIGs. 3A-3D show spacer length tunes pre-excision expression level sets range of activity of Digital Inputs, editing, And Logic (DIAL). FIGs. 3 A is a schematic of the editable promoter highlighting the spacer with variable length. In the plot, geometric mean fluorescence intensity (MFI) of mGL with different spacer in combination with ZFal, ZFa2, and Cre. Circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). Representative histograms of the mGL fluorescence intensity on the right sampled from bioreplicates (n=3). FIG. 3B shows plots of fold change which is the ratio of the -Cre condition to the +Cre condition for each spacer length. Circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 3C shows a schematic showing the PCR procedure for detection of promoter editing. FIG. 3D shows Cre- based editing efficiency as calculated from the gel by dividing the intensity of the edited band by the intensity of the unedited band.

[0042] FIGs. 4A-4D show single TF multimodal output. FIG. 4A is a schematic showing an example of a DIAL promoter with a single TF binding site for ZFla and a single spacer excisable by Cre or VCre. FIG. 4B is a schematic showing that the same recombinase recombination system can be used with a different zinc finger. FIG 4C is a schematic showing that different combinations of VCre and Cre and nested spacers, can lead to a diversity of expression states even with a single ZFa, due to corresponding multiple promoter states induced by recombinase-mediated DNA editing. FIG. 4D shows expression data in HEK293Ts that were transiently transfected cassettes containing promoters of various spacer lengths and mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), - / + Cre (11 ng), - / + Vcre (l ing) and - / + ZFa (14ng).

[0043] FIGs. 5A-5E show ZF titration: DIAL generates stable set points of expression across a large range of transactivator expression. FIG. 5A is a schematic of expected dosage curves with varying transactivator levels. FIG. 5B shows different levels of ZFla or ZF2a expression were achieved via a 2:1 serial dilution of an EFla-plasmid from IX (112 ng) to .00625X. Points on the scatter plot represent geometric means of independent biological replicates (B: n=4, C: n=6) for target gene (mGL) versus ZFla or ZF2a (mCherry or TagBFP, respectively) normalized based on the 0.125X ZFa and -Cre condition from each. FIG. 5C shows geometric means of the shown gated mGL expression from the ZFal or ZFa2 titration with three independent bioreplicates (n=3). Point represent geometric means of mGL, mCherry, or TagBFP from constructs with different strength promoters for ZF activator expression (112 ng), normalized based on the EFla, -Cre condition for ZF37, and the EFS, -Cre condition for ZF43. FIG. 5D is a schematic showing predicted effects of varying concentrations of a ZF1 inhibitor and a ZF1 activator on expression states. FIG. 5E shows different levels of ZFli expression were achieved via a 2:1 serial dilution of an CMV-plasmid.

[0044] FIGs. 6A-6D show user-defined, temporal control of DIAL via small-molecule regulation of recombinases and transactivators. Inducible recombinase and transactivators allow for temporal and user-defined control of expression levels. FIG. 6A shows data from HEK293Ts which were transiently transfected with cassettes with the 203bp-spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), each half of GIB- inducible Cre (56ng each), and - / + ZFa or empty plasmid vector (112.5ng), for a total of 450ng DNA per condition. Conditions had - / + luM GIB added at 1DPT. Conditions were flowed at 3DPT. Representative histograms sampled from bioreplicates (n=3) of combinations of ZF and GIB. Plotted circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 6B shows data from HEK293Ts which were transiently transfected with cassettes with the 203bp- spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), and - / + ZFa or empty plasmid vector (112.5ng), for a total of 338 ng DNA per condition. At 1DPT, cells were transfected with either 0, 350, or 500 ng of Cre modRNA. Conditions were flowed at 3DPT. Plotted circular points represent the fraction above 1500 fluorescence intensity for each bioreplicate, indicated cells that had been pushed into a higher expression regime. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 6C shows data from HEK293Ts which were transiently transfected with cassettes with the 203bp-spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), DOX-inducible ZFa (14ng), rtTA (28ng), and - / + Cre (ling). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). At 1DPT, DOX (1 ug / mL) was added, and cells were flowed at 3DPT. Plotted circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 6D shows data from HEK293Ts which were transiently transfected with cassettes with the 203bp- spacer TRE3G promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), rtTA (112.5ng), and - / + Cre (l ing). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). At 1DPT, DOX (IX = lug / mL and serially diluted) was added and cells were flowed at 3DPT. Plotted circular points represent the geometric mean of all cells in a single well (n=3).

[0045] FIGs. 7A-7C show DIAL generates programmable setpoints of transgene expression in primary cells. FIG. 7A shows the DIAL reporters are delivered to mouse embryonic fibroblasts (MEFs) via lentiviruses at 1 dpi. The inputs for activation (ZFal, ZFa2, and Cre) were delivered via retroviruses at days -1, and 0. Cells were flowed at three days post infected (dpi). FIG. 7B shows representative histograms sampled from independent bioreplicates (n=l to n=2) of target gene mGL fluorescence intensity with different spacer lengths, without and without ZFal or ZFa2, and with or without Cre. Data was gated on the co-infection marker iRFP670. The line represents the gate for mGL positive cells. FIG. 7C shows geometric MFI of the target gene (mGL) with different spacer lengths and combinations of viruses. Plotted circular points represent the geometric mean of all cells in each well of each bioreplicate (n=l or n=2). Dashed points represent arithmetic mean of the bioreplicates + / - standard error. Data was gated on the co-infection marker iRFP670 and the mGL positive population.

[0046] FIG. 8 is a schematic showing the extensible framework provided by DIAL for designing synthetic promoters that generate heritable setpoints of gene expression and perform across a range of cell types and delivery systems.

[0047] FIG. 9A-9I show exemplary uses of the DIAL system. FIG. 9A is a schematic of the DIAL promoter system. DIAL uses combinatorial inputs of synthetic zinc finger transcription factor (ZFa) and Cre recombinase to generate distinct setpoints of gene expression from a single promoter. FIG. 9B is a schematic of the pre-excision and post-excision state of the 203-base pair (bp) spacer DIAL promoter before and after Cre-mediated editing, respectively. The excision of the floxed 203 bp spacer increases expression by reducing the distance between the ZF binding sites and YB_TATA minimal promoter (bent arrow) from 316 bp to 79 bp. FIG. 9C is a logic table of inputs, ZFa and Cre, and outputs, expected promoter state, target setpoint, and observed gene (mGL) expression from the DIAL promoter. Output mGL single-cell distributions show output increase upon addition of ZFa (VP16-ZF37) and Cre with the 203 bp spacer DIAL promoter on plasmids intoHEK293T cells. Different combinations of inputs enable three setpoints. FIG. 9D is a representative fluorescence microscopy images of mGL expressed from 203 bp spacer DIAL promoter transfected with ZFa (VP16-ZF37) on plasmids into HEK293T cells, with or without Cre. Images taken 3 days post- transfection (dpt). FIG. 9E shows fold change of the output reporter expressed from DIAL promoters with different spacer lengths with co-transfected ZFa (VP16-ZF37) on plasmids into HEK293T cells, with or without Cre. Fold change is the output mGL geometric mean fluorescence intensity (MFI) normalized to the condition without Cre within each spacer length. Histograms show decreasing pre-excision expression for increasing spacer length, which generates the larger fold change upon addition of Cre. FIG. 9F shows fold change of the output reporter expressed from the 203 bp spacer DIAL promoter transfected with different zinc finger activators bearing different transactivation domains (ZF-TADs, e.g. ZFa) on plasmids into HEK293T cells, with or without Cre. Fold change is the output mGL geometric MFI normalized to the condition with VP16-ZF43 without Cre. Fold change increases with stronger ZFa. FIG. 9G shows a schematic of nested DIAL promoter with loxP and VloxP sites. Based on the combination of ZFa, VCre, and Cre, the nested DIAL promoter generates three promoter states and four different setpoints of expression. FIG. 9H is a logic table of inputs, ZFa, Cre, and VCre, and outputs, promoter state, target setpoint, and observed gene (mGL) expression for the nested DIAL promoter. Nested spacers enable four setpoints. FIG. 91 shows fold change of the output reporter expressed from the nested DIAL promoter transfected on plasmids into HEK293T cells, with or without ZFa, Cre or VCre. Fold change of output mGL geometric MFI is normalized to the condition with VP16- ZF43 without either recombinase.

[0048] FIG. 10 shows minimal promoter selection is an additional parameter for tuning DIAL setpoints. FIG. 10 (left panel) shows that output reporter mGL geometric MFI from 203 bpspacer DIAL promoters with different minimal promoters. Constructs were co-transfected into HEK293T cells with or without ZFa (VP16-ZF37) and Cre. The choice of minimal promoter influences the levels of pre- and post- excision expression, fold change, and basal activity. FIG. 10 (right panel) shows single-cell distributions of output mGL expressed from the 203 bp spacer DIAL promoters with different promoters according to the legend in FIG. 10 (left panel). Dotted histogram represents untransfected cells. The choice of minimal promoter also influences the shape of the single cell distributions.

[0049] FIGs. 11 A-C show minimal promoter selection is an additional parameter for tuning DIAL setpoints in a subset of tetracycline-inducible DIAL systems. FIG. 11A shows fold change of output reporter mGL fold change from 203 bp-spacer DIAL promoters with different minimal promoters. Constructs were co-transfected into HEK293T cells with or without Cre in the presence of DOX (1 ug / mL) and Cre. Fold change is the output mGL geometric MFI normalized to the condition without Cre within each minimal promoter. FIG. 1 IB shows output reporter mGL geometric MFI from 203 bp- spacer DIAL promoters with different minimal promoters. Constructs were co-transfected into HEK293T cells with or without Cre, and DOX (1 ug / mL) and Cre as according to the legend in FIG. 11 A. The choice of minimal promoter influences the levels of pre- and post- excision expression, and basal activity. FIG. 11C shows single-cell distributions of output mGL expressed from the 203 bp spacer DIAL promoters with different minimal promoters as according to the legend in FIG. 11 A. The choice of minimal promoter also influences the shape of the single cell distributions.

[0050] FIGs. 12A-12C show that DIAL promoter can be dually responsive to two smallmolecule inputs. FIG. 12A is a schematic of gibberellin (GIB)-inducible split Cre and DOX- inducible ZFa operably linked to a tetracycline-responsive element (TRE) promoter. FIG. 12B shows output reporter mGL geometric MFI from the 203 bp spacer DIAL promoter for mGL transfected with GIB inducible split Cre, rtTA, and TRE-VP16-ZF37-mCherry on plasmids into HEK293T cells. DOX (0.1 ug / m) turns expression “OFF” or “ON”, whereas presence of GIB (1 pM) determines levels of “ON” expression. FIG. 12C shows output mGL single cell distributions according to the experiment and legend in FIG. 12B. Dotted line represents single cell distribution for untransfected cells.

[0051] FIG. 13 shows: Left panel- a logic table of DOX and GIB inputs leading to three different output expression setpoints. Right panel- Output reporter mGL fold change from the 203 bp spacer DIAL promoter transfected with gibberellin (GIB) inducible split Cre, rtTA, and TRE-VP16-ZF37-mCherry on plasmids into HEK293T cells. Cells were treated with or without DOX (0.1 ug / m) and GIB (1 pM). Fold change is the mGL geometric MFI normalized to the condition with DOX and without GIB.

[0052] FIG. 14 shows output reporter mGL and input ZFa (VP16-ZF37-mCherry) single cell distributions using Flp, Bxbl, and PhiC31 recombinases. Output mGL is expression from 203 bp spacer DIAL promoter (112.5 ng) transfected with ZFa (112.5 ng) on plasmids into HEK293T, with or without recombinase (11 ng). Cells were gated on transfection control (iRFP670, 112.5ng). The 203 bp spacer was flanked by either F14 sites, Bxbl attB / attP sites (not shown), or PhiC31 attB / attP sites (not shown). Output mGL expression increases upon addition of cognate recombinase for each type of recognition site; comparable results were observed for each recombinase system. Input ZFa expression proxied by mCherry did not change with addition of cognate recombinase.

[0053] FIGs. 15A-15E show that DIAL frameworks extend tunable setpoints across transactivator systems. FIG. 15A is a schematic of TET-DIAL promoter system, depicting use of TET-DIAL combinatorial inputs of DOX and Cre along with co-delivery of rtTA to generate distinct setpoints of gene expression from a single promoter. FIG. 15B is a schematic of the preexcision and post-excision state of the TET-DIAL promoter before and after Cre-mediated editing, respectively. The excision of the floxed spacer reduces the distance between the tetO sites and minCMV minimal promoter. FIG. 15C shows output mGL single-cell distributions from the 610 bp spacer TET-DIAL promoter with rtTA transfected on plasmids into HEK293T cells, with and without Cre at different levels of DOX titration (1 ug / mL titrated down). Addition of Cre increases reporter expression, whereas titrating DOX results in concurrent changes in fraction of reporter positive cells and expression level. Gate is drawn to isolate cells with expression above the no DOX condition (lightest yellow). FIG. 15D shows output mGL geometric MFI versus DOX concentration for DOX titration shown in FIG. 15C for the 610 bp spacer TET-DIAL promoter. FIG. 15E shows fold changes of the output reporter mGL expressed from the TET-DIAL promoter of varying spacer lengths transfected with rtTA on plasmids into HEK293T cells with DOX (1 pg / mL). Fold change the output mGL geometric MFI normalized to the condition without Cre within each spacer length. Longer spacer lengths generate larger fold changes upon addition of Cre. Points in scatter plot D and summary plots represent mean of biological replicates with span indicating standard error(n=3). Points in scatter plot H represents individual bioreplicates (n=3). Histograms represent single-cell distributions sampled across bioreplicates (n=3).

[0054] Unless otherwise stated, units for output MFI are arbitrary units (a.u.), and fold change is unitless. Large markers represent the mean of biological replicates with span indicating standard error (n=3). Histograms represent single-cell distributions sampled across bioreplicates (n=3). Statistical significance was calculated with Students t-Test with ns p>0.05; *p<0.05; **p<0.01;

[0055] *** p<0.001.

[0056] DETAILED DESCRIPTION

[0057] Disclosed herein is a system capable of generating diverse modes of transgene expression from a single genetic founder. This system uses a combination of Digital Inputs, editing, And Logic (and is referred to hereinafter as “DIAL”) to generate programmable, unimodal setpoints of transgene expression. Using the DIAL system, expression may be tightly controlled and titrated across modes to, for instance, examine the dose-dependent impact on cellular phenotypes. As subtle changes in gene expression can generate diverging cell fates, the system disclosed herein provides a tool that allows fine-scale, unimodal tuning of gene expression. Importantly, as cell fates emerge over longer time scales, the DIAL systems provided herein can generate heritable changes in gene expression that can be recorded and read at terminal timepoints.

[0058] In some aspects, the DIAL systems provided herein are comprised in an engineered nucleic acid. In some embodiments, the engineered nucleic acid comprises a regulatory region operably linked to a gene (e.g., a transgene) or a site for gene insertion. As used herein, a regulatory region is any sequence which can regulate (e.g., promote, enhance, silence) expression or one or more genes (e.g., a transgene) or sites for gene insertion to which it is operably coupled. In some embodiments, an engineered nucleic acid comprises one or more regulatory regions operably linked to one or more genes (e.g., a transgene) or sites for gene insertion to which it is operably coupled. As used herein, “operably linked” regulatory regions are those which are capable of effecting or modulating expression of a gene (e.g., have the gene or site for gene insertion under transcriptional control). In some embodiments, a regulatory region (e.g., promoter) is upstream of a gene to which it is operably linked. As used herein, the term “gene” refers to a coding sequence. A coding sequence is a sequence of nucleotides which can, under certain conditions, be transcribed and / or translated to generate a payload. In some embodiments, a gene is a transgene. In some embodiments, a gene is transcribed and / or translated by transcription / translation machinery to generate a payload. As used herein, the term “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A payload may be a functional nucleic acid, a peptide, or a protein or fragment thereof.

[0059] In some embodiments, the regulatory region is a promoter. A “promoter” is a sequence in DNA which is recognized by the endogenous or introduced transcription machinery of a cell such that it directs the initiation of transcription of an RNA product from a gene. A promoter can be understood to be “operably linked to” a gene or have a gene “under transcriptional control” if it is in the correct location and orientation relative to the coding sequence such that it is capable of promoting expression (e.g., transcription) of the gene. Promoters may be native, constitutive, inducible, tissue specific, or synthetic. In some embodiments, a regulatory region is derived from a reference promoter (e.g., modified from a naturally occurring or synthetic promoter). In some embodiments, a regulatory region is derived from a naturally occurring promoter, e.g., is modified from a naturally occurring promoter. In some embodiments, a regulatory region is derived from a synthetic promoter. In some embodiments, a regulatory region is a minimal promoter. A “minimal promoter”, also referred to as a core promoter, is a region of DNA that serves as a scaffold for assembly of a pre-initiation complex (PIC), which is comprised of RNA polymerase II (Pol II), basal transcription factors and promoter DNA. Once assembled, PIC directs accurate transcription initiation by Pol II. In some embodiments, a minimal promoter comprises a transcription initiation site or portion thereof. In some embodiments, a minimal promoter comprises an entire transcription initiation site. In some embodiments, a minimal promoter comprises a portion of about 25-150bp (e.g., about 25-150bp, about 25-125bp, about 25-100bp, about 25-75bp, about 25-50bp, about 50-150bp, about 50-125bp, about 50-100bp, abut 50-75bp, about 75-150bp, about 75-125bp, about 75-100bp, about 100-150bp, about 100- 125bp, about 125bp-150bp) portion within a transcription initiation site. In some embodiments, a minimal promoter comprises a 50-100 bp portion within a transcription initiation site. In some embodiments, a minimal promoter comprises a transcription initiation site or portion thereof, and a 5’ flanking region, wherein the 5’ flanking region is 5’ of the transcription initiation site or portion thereof. In some embodiments, a minimal promoter comprises a transcription initiation site or portion thereof, and a 3’ flanking region, wherein the 3’ flanking region is 3’ of the transcription initiation site or portion thereof. In some embodiments, a minimal promoter comprises a transcription initiation site or portion thereof, a 5’ flanking region, and a 3’ flanking region. In some embodiments, the 5’ flanking region and the 3’ flanking region are the same length. In some embodiments, the 5’ flanking region and the 3’ flanking region are different lengths. In some embodiments, the 5’ flanking region and / or the 3’ flanking region comprises about l-150bp (e.g., about l-150bp, about l-125bp, about l-100bp, about l-75bp, about l-50bp, about l-25bp, about l-15bp, about l-10bp, about l-5bp, about 5-150bp, about 5-125bp, about 5- lOObp, about 5-75bp, about 5-50bp, about 5-25bp, about 5-15bp, about 5-10bp, about 15-150bp, about 15-125bp, about 15-100bp, about 15-75bp, about 15-50bp, about 15-25bp, about 25- 150bp, about 25-125bp, about 25-100bp, about 25-75bp, about 25-50bp, about 50-150bp, about 50-125bp, about 50-100bp, abut 50-75bp, about 75-150bp, about 75-125bp, about 75-100bp, about 100-150bp, about 100-125bp, about 125bp-150bp). Various minimal promoters are known in the art. Non-limiting examples of minimal promoters include: YB_TATA, adenovirus major late promoter (ML), Initiator (Inr) motif, pJB42CAT5 (a minimal promoter derived from the human junB gene), DPE motif, simian virus 40 (SV40), and minimal cytomegalovirus (CMV) promoters (e.g., CMV53, minCMV, minCMV2). Several of these promoter motifs have fixed positioning relative to a single, well-defined transcription start site. For example, the TATA-box motif is located about 30bp upstream of a single dominant transcription start site in focused core promoters. Another core promoter motif with a fixed position relative to transcription initiation is the Inr motif, which directly overlaps the transcription start site. The Inr is more abundant than the TATA but is not universal, and its consensus sequence differs.

[0060] Regulatory regions can be understood to comprise at least one transcription factor binding site and a transcription start site. As used herein, the term “transcription factor binding site (TFBS)” refers to a DNA sequence which is recognized by a corresponding transactivator; a TFBS may be any suitable TFBS known in the art (for example, see: Rauluseviciute I, el al. “JASPAR 2024: 20th anniversary of the open-access database of transcription factor binding profiles.” Nucleic Acids Res. in_press; doi: 10.1093 / nar / gkadl059). In some embodiments, a regulatory region comprises two or more TFBS. The inventors have found that, in contrast to concern in the art that increasing levels of transcription factors may influence the stability of promoter setpoints, transactivator levels above a threshold do not negatively impact DIAL system-mediated gene expression levels. As demonstrated in the Examples contained herein, DIAL systems generate stable setpoints that are robust to changing transactivator levels.

[0061] In some embodiments, a regulatory region comprises at least one transcription factor binding site. In some embodiments, a regulatory region comprises multiple transcription factor binding sites. In some embodiments, a regulatory region comprises multiple tessellated transcription factor binding sites. In some embodiments, a regulatory region comprises multiple distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, a regulatory region comprises multiple tessellated distinct transcription factor binding sites, each specific for a different transactivator. In some embodiments, a regulatory region comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator. The term “specific for” as used herein refers to an affinity between two or more entities. For instance, a transcription factor binding site is specific for a transactivator when the transactivator preferentially recognizes and interacts with or binds to the binding site relative to other binding sites. In some embodiments, a transactivator is a constitutive transcription factor. In some embodiments, the transactivator is a synthetic zinc finger transcription factor. In some embodiments, a transactivator is a small-molecule responsive transcription factor. For example, in some embodiments, the transactivator is a Tetracycline (TET) activated transactivator or a doxycycline (DOX) activated transactivator. As used herein, the term “transcription start site (TSS)” refers to a DNA sequence which is recognized by an RNA polymerase (e.g., complexed with one or more transcription factors) to initiate transcription of a complementary mRNA. A regulatory region may comprise any suitable TSS known in the art (for example, see: Suzuki, Ayako, et al. "DBTSS / DBKERO for integrated analysis of transcriptional regulation." Nucleic Acids Res. 46, no. DI (2018): D229-D238).

[0062] In some embodiments, a regulatory region comprises a spacer. As used herein, the term “spacer” refers to a non-coding sequence (i.e., a nucleotide sequence which is not a gene) separating a TFBS and TSS. In some embodiments, a regulatory region comprises two or more spacers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Typically, a spacer is between about 20 and 1000 nucleotides in length. In some embodiments, a spacer is 20-600, 20-650 or 20-900 nucleotides in length. As used herein, “about” means plus or minus 10% or less of the value indicated. For example, about 100 nucleotides in length means that the length can be between 90-110 nucleotides (inclusive). In some embodiments, a spacer is between about 20 to about 100, about 20 to about 200, about 20 to about 300, about 20 to about 400, about 20 to about 500, about 20 to about 600, about 20 to about 700, about 20 to about 800, about 20 to about 900, or about 20 to about 1000 nucleotides in length. In some embodiments, a spacer is about 20 to about 60, about 60 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1000 nucleotides in length. In some embodiments, a spacer is 20- 900, 25-650, 25-500, 50-900, 50- 650, 100-900, 100-650, 200-900, or 200-650 nucleotides in length. In some embodiments, a spacer is about 20 to about 40, about 70 to about 90, about 145 to about 165, about 190 to about 210, about 250 to about 275, about 370 to about 390, or about 600 to about 620 nucleotides in length. In some embodiments, a spacer is about 25 to about 35, about 75 to about 85, about 150 to about 160, about 195 to about 205, about 255 to about 270, about 375 to about 385, or about 605 to 615 nucleotides in length. In some embodiments, a spacer is 27, 79, 155, 203, 263, 380, or 610 nucleotides in length. In some embodiments, a spacer is 203, 380, or 610 nucleotides in length. The skilled artisan will appreciate that increasing the length of the spacer increases the distance between the TFBS and TSS of the regulatory region, thereby decreasing the baseline (e.g., pre-excision) level of expression of any gene(s) operably linked to the regulatory region.

[0063] In some embodiments, a regulatory region comprises a recombination regulation system. As used herein, the term “recombination regulation system” refers to one or more recombination sites inserted into a regulatory region in an orientation such that, in the presence of a cognate recombinase, recombination (e.g., excision) of a portion of the regulatory region can occur. Recombinases such as Cre are commonly used to induce expression from OFF to ON by excising a “stop” polyA signal flanked by loxP sites (“floxed”) or to excise a target gene coding sequence to knock down expression. These systems are useful since DNA-based changes are long-term and heritable even arising from transient Cre activation. Whereas most recombinase systems provide binary control, the DIAL systems developed herein utilize recombinase- mediated DNA editing such that regulatory region state may be recorded and gene expression may be finely tuned across physiologically-relevant regimes and tune between setpoints in between the ON / OFF. To construct a DIAL system which could be used to generate heritable setpoints of expression, recombinases are used to edit regulatory regions comprising “floxed” spacers of varying length.

[0064] It has been recognized herein that varying the length of spacers (e.g., by excising portions thereof) in regulatory regions can be useful to tune output range of expression of a gene or site for gene insertion operably linked to the regulatory region. For example, by reducing the distance between the TFBS and the TSS, the level of gene expression can be increased. Thus, in the system disclosed herein the spacer may be designed to be excisable (e.g., are “floxed”) so that expression of the recombinase excises the spacer and transgene expression increases. As illustrated in the Examples, increasing spacer length decreases output expression, while increasing the length of excisable portions of the spacer increases output dynamic range postexcision; accordingly, the skilled artisan will appreciate that the length of the spacer and excisable portions therein may be varied according to the desired effect on post-excision expression.

[0065] A “recombination site,” as used herein, refers to a DNA sequence or “site” which can be recognized by a cognate recombinase. Non-limiting examples of recombination sites include loxP (e.g., loxP, VloxP, SloxP) sites, FRT sites, F14 sites, attB / attP Bxbl sites, and attB / attP phiC31 sites. The term “recombinase”, as used herein, refers to an enzyme which can “recognize” a recombination site, i.e., mediate recombination of DNA at a recombination site or between recombination sites. In some embodiments, a recombinase is a tyrosine recombinase or a serine recombinase. Non-limiting examples of tyrosine recombinases include Cre recombinases (e.g., Cre, VCre, SCre), FLP recombinases. Non-limiting examples of serine recombinases include Bxbl recombinases and PhiC31 recombinases. A recombinase is considered “cognate” to a specified recombination site when it can recognize the site to mediate recombination; for example, a VCre recombinase is considered cognate to a VloxP site, but not cognate to an FRT site.

[0066] In some embodiments, a recombination regulation system is a Cre recombinase recombination regulation system. In some embodiments, the Cre recombinase system is a Cre- loxP recombinase system, a VCre- VloxP recombinase system, or an SCre-SloxP recombinase system. In some embodiments, the Cre recombinase recombination regulation system comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) loxP sites, one or more VloxP sites, and / or one or more SloxP sites. In some embodiments, a recombination regulation system is a Flp recombinase recombination regulation system. In some embodiments, the Flp recombinase system is a Flp- FRT recombinase system or a Flp-F14 recombinase system. In some embodiments, the Flp recombinase recombination regulation system comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) FRT sites and / or one or more F14 sites.

[0067] In some embodiments, a recombination regulation system is a Bxbl recombinase recombination regulation system. In some embodiments, the Bxbl recombinase recombination regulation system comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) Bxbl- attB sites and / or one or more Bxbl-attP sites.

[0068] In some embodiments, a recombination regulation system is a PhiC31 recombinase recombination regulation system. In some embodiments, the PhiC31 recombinase recombination regulation system comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) PhiC31- attB sites and / or one or more PhiC31-attP sites.

[0069] In some embodiments, a recombinase is small-molecule responsive (e.g., activated by a small molecule, such as gibberellin (GIB)).

[0070] In some embodiments, the recombination regulation system is comprised in the spacer of the regulatory region. In some embodiments, a recombination regulation system comprises recognition sites inserted into the spacer in such an orientation that, in the presence of a cognate recombinase, excision of at least a portion of the spacer can occur; for example, in some embodiments, the recognition sites flank the portion of the spacer that is excised upon contact with the recombinase. In some embodiments, the recombination regulation system comprises an excisable spacer. As used herein, the term “excisable spacer” refers to a spacer comprising a recombination regulation system wherein recognition sites flank the spacer in such an orientation that, in the presence of a cognate recombinase, the entire spacer is excised from the engineered nucleic acid occurs.

[0071] In some embodiments, a spacer comprises one or more nested excisable spacers. As used herein, the term “nested excisable spacer” refers to an excisable spacer comprised within another spacer (e.g., within another excisable spacer, within a non-excisable spacer). In some embodiments, a spacer comprises two or more nested excisable spacers, wherein each nested excisable spacer comprises orthogonal recombination sites (i.e., comprises two different recombination regulation systems), such that excision of each nested excisable spacer is independent of excision of another. As illustrated in the Examples provided herein, orthogonal recombinases may be useful to edit nested excisable spacers, generating multiple promoter states and expanding the number of possible set points from one TFBS. For example, a spacer may comprise a first nested excisable spacer and a second nested excisable spacer, wherein the first nested excisable spacer is flanked by loxP sites, and the second nested excisable spacer is flanked by VloxP sites. In the presence of Cre recombinase and absence of VCre recombinase, the first nested excisable spacer, but not the second nested excisable spacer, could be excised from the spacer. Thus, expression of an operably linked gene may be increased to a first level at a first time point by introducing VCre recombinase (thereby excising the first nested excisable spacer), then further increased at a second time point by introducing Cre recombinase (thereby excising the second nested excisable spacer). In some embodiments, a spacer comprises 2 or more nested excisable spacers. In some embodiments, a spacer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more excisable spacers. In some embodiments, a spacer comprises 2 or more orthogonal recombination sites (e.g., 2 or more recombination regulation systems). In some embodiments, a spacer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more orthogonal recombination sites.

[0072] Also contemplated herein are methods of increasing expression of a gene operably linked to a regulatory region in a cell by excising a portion of a spacer comprised in the regulatory region. In some embodiments, the method comprises contacting the cell with a recombinase cognate to recombination sites flanking the portion of the spacer to be excised.

[0073] Overall, the DIAL system(s) offers user-defined, temporal control of transgene expression via small-molecule responsive or modRNA-mediated expression. As summarized in FIG. 8, DIAL systems provide means for tuning transgene expression, e.g., via recombinase- mediated or transactivator-mediated alteration of regulatory region states. In a first example, a DIAL system regulatory region may contain one or more recombination regulation systems, such that contact of the regulatory region with a cognate recombinase results in the excision of a spacer or portion thereof from the regulatory region, thus reducing the distance between the TFBS and TSS, thereby altering the state of the regulatory region. In another example, the DIAL system regulatory region also contains a combination of two or more different TFBSs, each specific to a different transactivator (e.g., constitutive and / or small-molecule transactivator), such that contact of the regulatory region with one or more of the transactivators (e.g., at variable levels) further modulates expression of the transgene.

[0074] In addition, DIAL systems can be further customized, for example, by utilizing constitutive and / or small-molecule responsive transactivators according to a desired outcome. DIAL systems also provide a means for temporal induction of transgene expression. In a first example, a cell containing a DIAL system is contacted with a heterologous mRNA (e.g., chemically modified mRNA (modRNA)) encoding a recombinase at a desired timepoint, thereby inducing (e.g., activating, increasing) expression of the recombinase and, subsequently, changing the expression level of the transgene at the desired time point. In another example, a cell containing a DIAL system expresses a small molecule-responsive recombinase, such that the cell contains an inactive recombinase in the absence of the small molecule; contact of the cell with the small molecule at a desired time point would thus activate the recombinase, thereby inducing (e.g., activating, increasing) expression of the transgene at the desired time point. In yet another example, a cell containing a DIAL system expresses a small molecule-responsive transactivator, such that the cell contains an inactive transactivator in the absence of the small molecule; contact of the cell with the small molecule at the desired time point would thus activate the transactivator, thereby inducing (e.g., activating, increasing) expression of the transgene at the desired time point. In a further example, a cell containing a DIAL system expresses: (1) a transactivator responsive to a first small molecule; and (2) a recombination system comprising a recombinase responsive to a second small molecule; such that the cell contains an inactive transactivator in the absence of the first small molecule and an inactive recombinase in the absence of the second small molecule. Contact of the cell with the first small molecule (i.e., the small molecule that activates the transactivator) determines whether expression of a transgene is ON / OFF. In the presence of the first small molecule (i.e., the small molecule that activates the transactivator), contact of the cell with the second small molecule (i.e., the small molecule that activates the recombinase) would activate the recombinase, thereby changing expression level of the transgene at the desired time point. The first small molecule for the transactivator could be removed to reversibly shift between ON / OFF expression.

[0075] The skilled artisan will recognize that DIAL systems are compatible for use with various vectors, such as viral vectors (e.g., lentiviruses), and a variety of cell types (e.g., immortalized embryonic stem cells, primary embryonic stem cells, induced pluripotent stem cells). In some embodiments, a vector is a plasmid. In some embodiments, a vector a viral vector (e.g., a lentivirus, a retrovirus). In some embodiments, a transactivator is delivered via a vector (e.g., a plasmid, lentivirus, or retrovirus). Importantly, DIAL systems control gene expression in primary mouse embryonic fibroblasts, demonstrating the use for engineering primary cells for biomedicine and biomedical research. DIAL expands the mammalian cell engineering toolkit to achieve temporally dynamic gene expression profiles in vivo, without re-engineering the cell. Thus, DIAL can aid in precise control of transcription factors in cellular reprogramming, which can contribute to advances in cellular engineering for reprogramming, therapy, disease modeling, and more. By setting stable and heritable setpoints, DIAL provides a tool to investigate how transgene levels map to phenotypes. Overall, DIAL can be used to investigate how transgene levels influence cellular state, offering insights for cell engineering, disease modeling, and cell-based therapies.

[0076] EXAMPLES

[0077] Example 1: Promoter editing generates a range of unimodal setpoints from a synthetic promoter

[0078] In synthetic zinc-finger based promoter systems, the distance between the transcription factor binding sites (TFBS) and the transcription start site sets the level of expression. An excisable spacer was introduced to generate two distinct modes of expression from the pre- and post-excision states (FIG. 2A, FIGs. 9A-9C). Introduction of the tyrosine recombinase Cre excises the floxed spacer increasing expression in the presence of the cognate transcription factor as shown in FIG. 2B and FIG. 9B. Synthetic zinc-finger transcription factors (ZFa) are composed of a zinc-finger DNA-binding domain and the transcriptional activator. Different ZFas generate distinct maximal levels of expression and fold-changes. Using a single ZFa, the engineered promoter exhibits three distinct modes of expression, OFF, LOW, and HIGH - adding an additional setpoint to the typical OFF / ON from ZFa-inducible promoters. By constructing a synthetic promoter of tessellated binding sites that are recognized by different zinc-finger transcription factors (ZFal, ZFa2), we generate two setpoints of expression from each promoter state. By editing and choice of either ZFa, we can set five distinct modes of expression from a single founder construct. HEK293Ts were transiently transfected with the 203bp-spacer promoter with various target genes (112.5ng), transfection control (iRFP670, 112.5ng), - / + Cre (11 ng), and - / + ZFa (112ng). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). Cells were flowed at 3DPT and gated based on a transfection control. Logic table for how input ZFal and Cre combinations lead to output expected reporter states and experimentally different expression levels shown in histograms sampled from bioreplicates (n=3), gated by a cotransfected marker). Different inputs lead to different expression states, as shown in the logic gates of FIG. 9C. Per ZFa, addition of Cre leads to higher expression and in mGL matches the expression of the control cassette. (FIGs. 2C-2D). A representative fluorescence microscopy images of mGL expressed from 203 bp spacer DIAL promoter transfected with ZFa (VP16-ZF37) on plasmids into HEK293T cells, with or without Cre is provided in FIG. 9D.

[0079] To test if we could generate different modes, we transfected plasmids bearing our engineered promoter regulating the target gene reporter into human cells. To generate distinct states, we cotransfected combinations of plasmids expressing ZFal, ZFa2, and Cre (FIGs. 2C- 2D), and in effect constructed a logic gate where the output is level of expression. By adding different combinations, we could titrate expression of the reporter set states across over an order magnitude (FIG. 2D). In the presence of Cre and ZFas, expression from the post-edited promoter was similar to expression from a promoter lacking a spacer FIG. 2D). Importantly, each setpoint showed a single mode of expression that corresponds to the molecular state programmed by the combination of inputs (FIG. 2C). By tagging each ZFa with a fluorescent protein, we verified that the levels of ZFa do not change in response to Cre. Therefore, expression differences are attributable to activity of Cre, not difference in ZFa levels. Upon Cre-mediated excision of the spacer, we expect that the distance between the binding sites and minimal promoter will decrease and that this causes the change in expression. To verify Cre-mediated promoter editing, we extracted DNA and amplified the promoter region via PCR (FIG. 2F). As expected, in the presence of Cre, a shorter band appears, corresponding to the edited promoter. We observed incomplete editing, which may contribute to how while we observe mostly unimodal trends, the slight deviations in unimodal expression at higher expression likely reflect incomplete editing (FIG. 2C).

[0080] In addition to Cre-loxP based system, various recombinase systems, such as Flp, Bxbl, and PhiC31, can be used with DIAL, e.g., to excise spacers and alter expression. DIAL frameworks were created with other recombinase systems, using analogous designs of the 203 bp spacer flanked by loxP sites. 203bp spacers were flanked with: F14 sites cognate to Flp tyrosine recombinase; attB / attP sites cognate to Bxbl recombinase; or attB / attP sites cognate to PhiC31 recombinase. All recombinase sites were oriented in the same direction. As shown in FIG. 14, DIAL promoters with excisable spacers can be prepared using Flp, Bxbl, and PhiC31 recombinases. In all cases, we demonstrated an increase of expression with addition of the cognate recombinase.

[0081] DIAL is an expandable tool for controlling any target gene. We demonstrate that DIAL generates unimodal setpoints of transgenes encoding tagged fusions of human and mouse genes including p53-Halo and mCherry-HRas. DIAL can also be used to regulate multiple genes from polycistronic cassettes (FIG. 2F). Altogether, the DIAL promoter system generates two distinct promoter states and five unimodal expression levels that can be “dialed” to defined, stable setpoints. Students t-Test with ns p>0.05; *p<0.05; **p<0.01; *** p<0.001

[0082] Example 2: Changing spacer length tunes pre-excision setpoint expression level

[0083] To explore how promoter activity was affected by the distance of the TFBS array to the transcription start site, we varied the length of the floxed spacer. While the initial distance to the promoter varied by length of the floxed spacer, the edited promoter and static control retain 79 bp between the TFBS array and the minimal core promoter. We expected that the unedited promoters would show lower expression as spacer length increases. With the addition of Cre, we expected that edited constructs would converge to a similar level of expression.

[0084] From both the summary of the geometric mean fluorescence activity (MFI) and the flow plots (FIGs. 3A-3B), we found that as expected increasing spacer length reduces expression of pre-excision promoters for both ZFal and ZFa2 (FIGs. 3A-3B). The synthetic promoter showed greatest sensitivity for spacers between 79 to 203 bp with limit effects at smaller and larger spacer lengths. Curiously, we noticed that addition of Cre slightly reduced expression of the control, suggesting Cre may interfere to limit maximal expression. Nonetheless, as expected, expression from the post-excision state of the various promoters converges to the control. By reducing expression of the pre-excision state, increasing space length increases the range and fold-change between the setpoints, allowing tuning to different intermediate levels (FIGs. 3C- 3D, FIG. 9E).

[0085] FIGs. 3A-3F, 9E show spacer length tunes pre-excision expression level sets range of activity of DIAL. HEK293Ts were transiently transfected cassettes containing promoters of various spacer lengths and mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), - / + Cre (11 ng), and ZFa (14ng). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). All conditions were flowed at 3DPT and gated based on the transfection control. (3 A, 3B): Schematic of the editable promoter highlighting the spacer with variable length. In the plot, geometric mean fluorescence intensity (MFI) of mGL with different spacer in combination with ZFal, ZFa2, and Cre. Circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). Representative histograms of the mGL fluorescence intensity on the right sampled from bioreplicates (n=3). (3C, 3D, 9E) Plots of fold change which is the ratio of the -Cre condition to the +Cre condition for each spacer length. Circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). (3E) The schematic shows the PCR procedure for detection of promoter editing. The gel shows bands from PCR of technical replicates of cell lysate samples with ZFla - / + Cre. Cre leads to spacer excision of the editable promoter, although not completely efficient. FIG. 3G shows Cre-based editing efficiency as calculated from the gel by dividing the intensity of the edited band by the intensity of the unedited band.

[0086] Deviations in unimodality at short spacer lengths or non- significant difference in expression for the 27bp spacer may reflect a diversity of promoter-edited states that results from inefficient excision of the spacer. To examine this hypothesis, we used PCR to amplify the reporter region isolated from cells pre- and post-excision via Cre (FIG. 3E). We find that the two shortest spaces achieve lower rates of excision while the 203 bp spacer achieves optimal excision (FIG. 3F). Using only a single ZFa we find that editing the distance between the binding sites and the minimal promoter changes expression. Additionally, the length of spacer changes both the absolute values of the pre-excision setpoints and thus the fold changes between pre- and post-excision states.

[0087] Indeed, as shown in FIG. 9E, HEK293T cells transfected with DIAL promoters operably linked to a reporter gene and co-transfected with ZFa (VP16-ZF37) on plasmids, pre-excision expression decreases with increasing spacer lengths. DIAL promoters with increased spacer generated a larger fold change in expression of the reporter genes upon addition of Cre, indicating that variable spacer length can be used to tune expression precisely.

[0088] ZFa strength was also found to affect reporter expression. Cells containing DIAL promoters operably linked to a reporter gene were transfected with different zinc finger activators bearing different transactivation domains (ZF-TADs, e.g. ZFa) on plasmids into HEK293T cells, with or without Cre. Fold change is shown in FIG. 9F as the output mGF geometric MFI normalized to the condition with VP16-ZF43 without Cre. As shown in FIG. 9F, fold change in reporter expression increases with stronger ZFa, as well as the pre-excision and post-excision setpoints.

[0089] Example 3: Nested, excisable spacers expand the number of setpoints

[0090] To expand the number of setpoints possible from a single transactivator, we sought to build a nested set of excisable spacers. By excising spacers of different lengths, we can set multiple levels of expression based on the distance of the TFBS to the core promoter using only a single ZFa (FIGs. 4A-4B, FIG. 9G). To this end, we encoded a set of nested spacers. By flanking the spacers with orthogonal recombination sites, loxP and VloxP, recognized by different cognate tyrosine recombinases, Cre and VCre, respectively, we could selectively define the expression setpoint based on the specific combination of ZFa and recombinase (FIGs. 4C- 4D, FIG. 9H). We flanked the internal spacer VloxP sites and flanked the outer spacer loxP. Addition of VCre excises the internal spacer leading to an intermediate spacer length. Addition of Cre, excises the entire space length, leading to the shortest distance and highest expected expression.

[0091] We tested the synthetic promoter with the nested, excisable spacer in transient transfection (FIG. 4E, FIGs. 9H-9I). Through addition of specific recombinases, each ZFa generates three defined setpoints (FIGs. 4E-F, FIGs. 9H-9I). Through combination of ZFa and recombinase, DIAE allows seven setpoints over a 15-fold range of expression, allowing for fine- scale titration of transgene expression.

[0092] FIGs. 4A-4C and 9G-H show multimodal output of different combinations of ZFa and recombinases. FIG. 4A and FIGs. 9G-H are schematics showing the nested spacers flanked by VloxP and loxP. Different combinations of VCre, Cre, and ZFa can lead to a diversity of expression states due to corresponding multiple promoter states induced by recombinase- mediated DNA editing (FIGs. 4B and FIGs. 9H-I). HEK293Ts were transiently transfected cassettes containing promoters of various spacer lengths and mGF target gene (112.5ng), transfection control (iRFP670, 112.5ng), - / + Cre (11 ng), - / + Vcre (l ing) and - / + ZFa (14ng). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (360 ng). All conditions were flowed at 3DPT and gated based on the transfection control. Plotted circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). FIG. 4C and FIG. 9H are representative histograms of the mGL fluorescence intensity on the right sampled from bioreplicates (n=3).

[0093] FIG. 91 shows fold change of the output reporter expressed from the nested DIAL promoter transfected on plasmids into HEK293T cells, with or without ZFa, Cre or VCre. Fold change of output mGL geometric MFI is normalized to the condition with ZFas having VP 16- ZF43 TADs without either recombinase.

[0094] Example 4: DIAL generates stable setpoints that are robust to changing levels of ZFa

[0095] Within specific regimes, synthetic promoters increase expression in response to the levels of transactivators. Given that binding arrays composed of multiple binding sites show strong cooperativity, we expected that for increasing expression of ZFa the mean expression would follow a Hill function (FIG. 5A). Similar to other synthetic promoter systems, at low levels of ZFa, cells exist in a bimodal regime, where cells distribute between “OFF” and “ON” states (FIG. 5B). Above a threshold of ZFa expression, expression from the synthetic promoter becomes invariant to the levels of ZFa, demonstrating a stable mode of expression that depends on the combination of ZFa and Cre inputs (FIG. 5 A).

[0096] Given this responsiveness to ZFa, our editable promoter allows transit between two versions of these titration curves. To explore this relationship between ZFa and expression, we delivered different doses of plasmids to change the levels of ZFa. We could measure via the associated fluorescent protein reporter for each ZFa (FIGs. 5C-5D, 5F-5G). As we expected, excision of the spacer via Cre addition allows us to transit between stable points along the titration curves (FIG. 5C, 5F). At high levels of ZFa, the synthetic promoter system is in a dosage-invariant regime (FIGs. 5C-5D, 5F-5G). At lower levels of ZFa, we observe the expected biomodal regime, but where the higher mode matches the peak of the dosage invariant region (FIG. 3A-C). The biomodality contributes to the lower means in the titration curve, but the dosage invariance of the higher mode is seen after gating (FIGs. 5E, 5H).

[0097] To explore the generality of dosage invariance, we generated a range of ZFa expression using a panel of constitutive promoters of different strengths (FIGs. 5E, 5H). Across the range of ZFa expression, expression from the synthetic promoter maintained stable expression in the dosage-invariant regime. Together, we find that DIAL platform offers both flexibility and robustness in generating stable setpoints of transgene expression.

[0098] To introduce some ability to tune each stable setpoint, we utilized a zinc finger inhibitor (ZFi). The ZFi binds to the same binding sites as ZFal, but is linked to dsRed instead of the VP 16 activator domain.2 The competition for the binding sites modulates the amount of expression (FIG. 51). With constant ZFa expression, higher ZFi expression led to a decrease in expression (FIGs. 5J-5K). The titration curves with and without Cre were consistently separated across ZFi levels, demonstrating the ability to use this system to toggle between titration curves.

[0099] FIGs. 5A-5E show ZF titration: DIAL generates stable set points of expression across a large range of transactivator expression. HEK293Ts were transiently transfected with the 203bp- spacer promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), - / + Cre (11 ng), and varying amounts of ZFa or ZFi (0 to 112ng). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). All conditions were flowed at 3DPT and gated based on the transfection control. (FIG 5A): Schematic of expected dosage curves with varying transactivator levels. (FIG. 5B): Different levels of ZFla or ZF2a expression were achieved via a 2:1 serial dilution of an EFla-plasmid from IX (112 ng) to .00625X. Points on the scatter plot represent geometric means of independent biological replicates (B: n=4, C: n=6) for target gene (mGL) versus ZFla or ZF2a (mCherry or TagBFP, respectively) normalized based on the 0.125X ZFa and -Cre condition from each. (FIG. 5C) Gray and black points represent geometric means of the shown gated mGL expression from the ZFal or ZFa2 titration with three independent bioreplicates (n=3). Points represent geometric means of mGL, mCherry, or TagBFP from constructs with different strength promoters for ZF activator expression (112 ng), normalized based on the EFla, -Cre condition for ZF37, and the EFS, -Cre condition for ZF43. (FIG. 5D) Different levels of ZFli expression were achieved via a 2:1 serial dilution of an CMV-plasmid. Across this serial dilution, ZFla was constant at 0.5x (56ng). Points represent geometric means of independent bioreplicates (n=4) of the target gene (mGL) versus the ZFli (dsRed), normalized by the OX ZFli, -Cre condition. (5E) Representative histograms of varying levels of the ZFli plasmid titration and the corresponding target gene expression with or without Cre.

[0100] Example 5: User-defined temporal of expression with DIAL By interplaying with inducible tools for transactivator or Cre expression, we can use DIAL externally induce expression changes at a user-defined timepoint. We demonstrated responsiveness of DIAL to gibberellin (GIB) inducible Cre (FIG. 6A), and also modRNA- delivered Cre (FIG. 6B) (Weinberg, B. H. et al. High-performance chemical- and. light-inducible recombinases in mammalian cells and mice. Nat Commun 10, 4845 (2019).). The modRNA can be preferable because it does not require the prior delivery of additional genetically encoded parts prior to its use. Both these methods exhibited greater inefficiency than constitutive Cre, and could be further optimized for delivery and efficiency.

[0101] On the transactivator side, we encoded the ZFa to a DOX-responsive promoter. Upon addition of doxycline (DOX) ZFa expression started and target gene expression from the promoter began (FIG. 6C). We found that as before, the induction of Cre toggled expression from a low to high setpoint. However, there was lower expression in general when using TRE- ZFa, which we hypothesize arose from resource burden from the additional genetic parts. Therefore, DIAL can be used for temporal control of expression from the same construct. We next explored whether DIAL can be extended to another inducible-transactivator system.

[0102] Example 6: DIAL can be extended to the TET transactivator system

[0103] There are other transactivator systems that are abundantly used for inducible control. We explored the effect of inserting a floxed spacer in between TFBS for rtTA and the minimal YB_TATA promoter. Indeed, we found that, like before, upon the addition of Cre expression increased, especially in the highest levels of DOX induction (FIG. 6D). At lower levels, expression was similar, but this may be due to the clear bimodality of the expression system.

[0104] FIGs. 6A-6D show user-defined, temporal control of DIAL via small-molecule regulation of recombinases and transactivators. Inducible recombinase and transactivators allow for temporal and user-defined control of expression levels. (FIG. 6A) HEK293Ts were transiently transfected with cassettes with the 203bp-spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), each half of GIB-inducible Cre (56ng each), and - / + ZFa or empty plasmid vector (112.5ng), for a total of 450ng DNA per condition. Conditions had - / + luM GIB added at 1DPT. Conditions were flowed at 3DPT. Representative histograms sampled from bioreplicates (n=3) of combinations of ZF and GIB. Plotted circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). (FIG. 6B) HEK293Ts were transiently transfected with cassettes with the 203bp-spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), and - / + ZFa or empty plasmid vector (112.5ng), for a total of 338 ng DNA per condition. At 1DPT, cells were transfected with either 0, 350, or 500 ng of Cre modRNA. Conditions were flowed at 3DPT. Plotted circular points represent the fraction above 1500 fluorescence intensity for each bioreplicate, indicated cells that had been pushed into a higher expression regime. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). (FIG. 6C) HEK293Ts were transiently transfected with cassettes with the 203bp-spacer ZF-responsive promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), DOX-inducible ZFa (14ng), rtTA (28ng), and - / + Cre (l ing). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). At 1DPT, DOX (1 ug / mL) was added, and cells were flowed at 3DPT. Plotted circular points represent the geometric mean of all cells in a single bioreplicate. Dashed points represent arithmetic mean of the bioreplicates + / - standard error (n=3). (FIG. 6D) HEK293Ts were transiently transfected with cassettes with the 203bp-spacer TRE3G promoter with mGL target gene (112.5ng), transfection control (iRFP670, 112.5ng), rtTA (112.5ng), and - / + Cre (l ing). All conditions were filled with an empty plasmid vector to achieve the same amount of total DNA per condition (349 ng). At 1DPT, DOX (IX = lug / mL and serially diluted) was added and cells were flowed at 3DPT. Plotted circular points represent the geometric mean of all cells in a single well (n=3). Students t-Test with ns p>0.05; *p<0.05; **p<0.01

[0105] FIGs. 15A-15E show that DIAL frameworks extend tunable setpoints across transactivator systems. FIG. 15A depicts use of TET-DIAL combinatorial inputs of DOX and Cre along with co-delivery of rtTA to generate distinct setpoints of gene expression from a single promoter. FIG. 15B depicts predicted pre-excision and post-excision state of the TET- DIAL promoter before and after Cre-mediated editing, respectively, wherein excision of the floxed spacer reduces the distance between the tetO sites and minCMV minimal promoter. As shown in FIGs. 15C-E, addition of Cre increases output expression up to five-fold over preexcision levels across a range of spacer lengths (203bp, 380bp, and 610bp), with addition of Cre increasing reporter expression and titration of DOX resulting in concurrent changes in fraction of reporter positive cells and expression level. Example 7: DIAL controls gene expression in primary mouse embryonic fibroblasts

[0106] One application where DIAL can be useful is in the control of long cell processes including direct conversion of primary cells. If we can integrate a transcription factor or oncogene of interest, changing its levels at a user-defined timepoint in a way that guides cells through the desired trajectory. Due to the heritable nature of DNA level modifications, such encoded changes of expression via DIAL will be stable from a transient stimulus.

[0107] DIAL provides a tool for screening libraries of ORFs to identify genes that exhibit specific profiles of dosage-response on cellular phenotype. By generating a mixed population of edited and non-edited promoters, we can screen for which level low or high (or more with the nested spacer) leads to the desired phenotype. In the desired phenotype we can further perform PCR or sequencing to determine the relative enrichment or depletion of recombinase-mediated editing in specific subpopulation and phenotypes.

[0108] To examine DIAL in normal primary cells, we delivered the editable promoter via lentivirus into mouse embryonic fibroblasts (MEFs). Addition of the Cre recombinase increased expression. As expected from prior experiments in 293Ts, the longer spacer displayed a larger dynamic range, and we observed that addition of recombinase to the loxP controls induced as decrease in expression FIG. 7B-7C.) These results show that DIAL works in a virally integrated context, that Cre can be delivered via virus, and that the design principles behind DIAL work in primary cells as well.

[0109] FIGs. 7A-7C show DIAL generates programmable setpoints of transgene expression in primary cells. (7 A) The DIAL reporters are delivered to mouse embryonic fibroblasts (MEFs) via lentiviruses at 1 dpi. The inputs for activation (ZFal, ZFa2, and Cre) were delivered via retroviruses at days -1, and 0. Cells were flowed at three days post infected (dpi). (7B) Representative histograms sampled from independent bioreplicates (n=l to n=2) of target gene mGL fluorescence intensity with different spacer lengths, without and without ZFal or ZFa2, and with or without Cre. Data was gated on the co-infection marker iRFP670. The line represents the gate for mGL positive cells. (7C) Geometric MFI of the target gene (mGL) with different spacer lengths and combinations of viruses. Plotted circular points represent the geometric mean of all cells in each well of each bioreplicate (n=l or n=2). Dashed points represent arithmetic mean of the bioreplicates + / - standard error. Data was gated on the co- infection marker iRFP670 and the mGL positive population. Students t-Test with ns p>0.05; *p<0.05; **p<0.01; *** p<0.001

[0110] Example 8: Editable DIAL and small molecule-inducible DIAL promoter architectures are extendable to other minimal promoters

[0111] In this Example, DIAL systems were prepared with one of a variety of minimal promoters downstream of a ZFa binding site and 203-bp spacer. Tested promoters included MLP, pJB42CAT5, SV40, CMV53, and both a small and large version of minCMV (minCMV and minCMV2 respectively). As shown in FIG. 10, each unique DIAE system demonstrated expression increases upon addition of Cre for excision of the 203-bp spacer. These results indicate the DIAE framework is extensible to various minimal promoters. Further, as shown with tetracycline (TET) inducible DIAL systems in FIGs. 11A-11C, minimal promoters are suitable for use in small molecule-inducible DIAL systems.

[0112] Overall, minimal promoter choice offers another aspect of tunability to the system. The choice of minimal promoter influences the levels of pre- and post- excision expression, fold change, basal activity, as well as the shape of the single cell distributions. Further, as the exact cell type for any given application may vary, different promoters may be useful for different cell types.

[0113] Example 9: DIAL promoters can be dually responsive to two small molecules

[0114] In this Example, DIAL systems with small molecule-inducible ZFa and Cre components were prepared. As shown in FIG. 12A, a DIAL system comprising a doxycycline (DOX)- inducible ZFa encoded by a TRE-promoter, operably linked with a gene encoding mCherry was prepared and combined with a gibberellin (GIB)-inducible split Cre. The dual DOX / GIB system was predicted to induce expression according to the logic table in FIG. 13 (left panel). As shown in FIGs. 12B-12C and FIG. 13 (right panel), no expression was observed in the absence of DOX. While some expression was observed with DOX alone, DOX in combination with GIB achieved 3x expression (FIG. 12C).

[0115] Accordingly, DIAL systems can be prepared with independently inducible transactivators and recombination regulation systems, allowing for external control of output level via different combinations of small molecules.

Claims

CLAIMSWhat is claimed is:

1. An engineered nucleic acid, comprising: a regulatory region comprised of one or more transcription factor binding sites upstream of a transcription start site and separated by a spacer, wherein the one or more transcription factor binding sites are specific to one or more transactivators, wherein the regulatory region is upstream of a gene or a site for gene insertion, and wherein the regulatory region comprises: a recombination regulation system for excising at least an excisable portion of the spacer.

2. The engineered nucleic acid of claim 1, wherein the excisable portion of the spacer is at least 25 nucleotides in length.

3. The engineered nucleic acid of claim 1 or 2, wherein the spacer comprises nested excisable spacers and orthogonal recombination sites.

4. The engineered nucleic acid of any one of claims 1-3, wherein the recombination regulation system is a tyrosine recombinase or serine recombinase recombination system.

5. The engineered nucleic acid of claim 4, wherein the recombination regulation system comprises at least two recombination sites in the spacer.

6. An engineered nucleic acid, comprising: a regulatory region comprised of one or more transcription factor binding sites upstream of a transcription start site and separated by a spacer, wherein the one or more transcription factor binding sites are specific to one or more transactivators, wherein the regulatory region is upstream of a gene or a site for gene insertion, andwherein the spacer comprises nested excisable spacers and orthogonal recombination sites.

7. The engineered nucleic acid of claim 3 or 4, wherein each of the nested excisable spacers is at least 25 nucleotides in length.

8. The engineered nucleic acid of claim 6 or 7, wherein the spacer comprises 2-10 nested excisable spacers and 2-10 orthogonal recombination sites.

9. The engineered nucleic acid of any one of claims 1-8, wherein the regulatory region further comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator.

10. The engineered nucleic acid of claim 6, wherein the transactivator is a synthetic zinc finger transcription factor.

11. The engineered nucleic acid of claim 6, wherein the transactivator is a small moleculeinducible transactivator.

12. A method of regulating gene expression, comprising, contacting a cell with an engineered nucleic acid of any one of claims 1-11, wherein the engineered nucleic acid comprises a gene downstream of the regulatory region, and contacting the cell with a recombinase cognate to the recombination sites or a transactivator cognate to the transcription factor binding site to regulate expression of the gene.

13. The method of claim 12, wherein the transactivator is a vector encoding a transactivator protein.

14. The method of claim 13, wherein the transactivator protein is a synthetic zinc finger transcription factor.

15. The method of any one of claims 12-14, wherein the level of transactivator delivered to the cell is greater than a threshold level to achieve bimodal gene expression.

16. The method of any one of claims 13-15, wherein the vector encoding the transactivator protein comprises a constitutive promoter or an inducible promoter.

17. The method of any one of claims 12-16, further comprising contacting the cell with a transcription factor inhibitor.

18. The method of claim 17, wherein the transcription factor inhibitor is a zinc finger inhibitor.

19. The method of any one of claims 12-18, wherein the cell is a primary cell.

20. The method of any one of claims 12-18, wherein the cell is in a subject.

21. The method of any one of claims 12-20, wherein the engineered nucleic acid is incorporated in a lentiviral vector.

22. A viral vector, comprising, an engineered nucleic acid of any one of claims 1-11.

23. The vector of claim 22, wherein the viral vector is a lentivirus.

24. A method of increasing expression of a gene in a cell, the method comprising excising at least a portion of a spacer in a regulatory region of a nucleic acid of the cell, wherein the regulatory region comprises a transcription factor binding site and a transcription start site separated by the spacer, wherein the regulatory region is upstream of the gene, and wherein excising the portion of the spacer increases expression of the gene.

25. The method of claim 24, wherein excising the portion of the spacer comprises contacting the cell with a recombinase cognate to recombination sites flanking the portion of the spacer.

26. The method of claim 25, wherein the recombinase is a small molecule-inducible recombinase.

27. The method of any one of claims 24-26, wherein the regulatory region further comprises at least two tessellated distinct transcription factor binding sites, each specific for a different transactivator.

28. The method of any one of claims 24-27, further comprising contacting the cell with a transactivator.

29. The method of claim 28, wherein the transactivator is a vector encoding a transactivator protein.

30. The method of claim 29, wherein the vector encoding the transactivator protein comprises a constitutive promoter or an inducible promoter.

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