Abstract
The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions1,2,3,4,5,6,7,8,9,10,11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12,13,14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility6,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.
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Acknowledgements
We thank C. Jennings for comments, F. A. Ran for help with illustrations, C. Lin for editing, M. M. Cunniff for technical assistance and W. Yan for computational analysis, and members of the Zhang laboratory for discussion, support and advice. S.K. is supported by a Hubert Schoemaker Fellowship from the McGovern Institute for Brain Research at MIT. M.H. is supported by a postdoctoral fellowship from the Human Frontiers Science Program. G.M.C. is support by a NIH NHGRI CEGS grant (P50-HG005550). F.Z. is supported by a NIH Transformative R01 award (R01-NS073124), a NIH Director’s Pioneer Award (DP1-MH100706), the Keck, McKnight, Vallee, Damon Runyon, Searle Scholars, Klingenstein, and Simons Foundations, Bob Metcalfe and Jane Pauley. Sequence, protocol, and reagent information are available through the Zhang laboratory website at http://www.genome-engineering.org.
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Contributions
S.K., M.D.B. and F.Z. developed the concept and designed experiments. S.K., M.D.B., A.E.T., P.D.H., M.H. and D.A.S. carried out LITE-related experiments and analysed data. L.C. and P.D.H. developed the SID4X effector domain, the P11-targeting TALEs and the abscisic acid induction system. R.J.P. developed the Cas9 transcription activator and repressor systems. S.K., A.E.T., M.D.B., P.D.H. and F.Z. wrote the manuscript with input from M.H., L.C. and G.M.C.
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A patent application has been filed relating to this work, and the authors plan on making the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org).
Extended data figures and tables
Extended Data Figure 1 RNA-guided DNA binding protein Cas9 can be used to target transcription effector domains to specific genomic loci.
a, The RNA-guided nuclease Cas9 from the type II Streptococcus pyogenes CRISPR/Cas system can be converted into a nucleolytically inactive RNA-guided DNA binding protein (Cas9**) by introducing two alanine substitutions (D10A and H840A). Schematic showing that a synthetic guide RNA (sgRNA) can direct Cas9**-effector fusion to a specific locus in the human genome. The sgRNA contains a 20-bp guide sequence at the 5′ end which specifies the target sequence. On the target genomic DNA, the 20-bp target site needs to be followed by a 5′-NGG PAM motif. b, c, Schematics showing the sgRNA target sites in the human KLF4 and SOX2 loci, respectively. Each target site is indicated by the blue bar and the corresponding PAM sequence is indicated by the magenta bar. d, e, Schematics of the Cas9**–VP64 transcription activator and SID4X–Cas9** transcription repressor constructs. f, g, Cas9**–VP64- and SID4X–Cas9**-mediated activation of KLF4 and repression of SOX2, respectively. All mRNA levels were measured relative to GFP mock-transfected 293FT cells (mean ± s.e.m.; n = 3 biological replicates).
Extended Data Figure 2 Engineering of light stimulation parameters and activation domains of LITEs.
a, Illustration of the absorption spectrum of CRY2 in vitro. Cryptochrome 2 was optimally activated by 350–475 nm light23. A sharp drop in absorption and activation was seen for wavelengths greater than 480 nm. Spectrum was adapted from ref. 23. b, Impact of illumination duty cycle on LITE-mediated gene expression. Varying duty cycles (illumination as percentage of total time) were used to stimulate 293FT cells expressing LITEs targeting the KLF4 gene, to investigate the effect of duty cycle on LITE activity. KLF4 expression levels were compared to cells expressing GFP only. Stimulation parameters were: 466 nm, 5 mW cm−2 for 24 h. Pulses were performed at 0.067 Hz with the following durations: 1.7% = 0.25 s pulse, 7% = 1 s pulse, 27% = 4 s pulse, 100% = constant illumination. (mean ± s.e.m.; n = 3–4 biological replicates.) c, The transcriptional activity of CRY2PHR/ CIB1 LITE was found to vary according to the intensity of 466 nm blue light. Neuro 2a cells were stimulated for 12 h at a 7% duty cycle (1 s pulses at 0.067 Hz). All Neurog2 mRNA levels were measured relative to cells expressing GFP only (mean ± s.e.m.; n = 3–4 biological replicates). d, Light-induced toxicity measured as the percentage of cells positive for red-fluorescent ethidium homodimer-1 versus calcein-positive cells (mean ± s.e.m.; n = 3 biological replicates; **P < 0.01). e, We compared the activation domains VP16 and p65 in addition to VP64 to test the modularity of the LITE CIB1–effector component. Neurog2 upregulation with and without light by LITEs using different transcriptional activation domains (VP16, VP64 and p65). Neuro 2a cells transfected with LITE were stimulated for 24 h with 466 nm light at an intensity of 5mW cm−2 and a duty cycle of 7% (1 s pulses at 0.067 Hz). All three domains produced a significant light-dependent Neurog2 mRNA upregulation (P < 0.001). We selected VP64 for subsequent experiments due to its lower basal activity in the absence of light-stimulation (mean ± s.e.m.; n = 3–4 biological replicates).
Extended Data Figure 3 Chemical induction of endogenous gene transcription.
a, Schematic showing the design of a chemical inducible two-hybrid TALE system based on the abscisic acid (ABA) receptor system. ABI and PYL dimerize upon the addition of ABA and dissociate when ABA is withdrawn. b, Time-course of ABA-dependent Neurog2 upregulation. 250 μM of ABA was added to Neuro 2a cells expressing TALE(Neurog2)–ABI and PYL–VP64. Fold mRNA increase was measured at the indicated time points after the addition of ABA. c, Decrease of Neurog2 mRNA levels after 24 h of ABA stimulation. All Neurog2 mRNA levels were measured relative to GFP-expressing control cells (mean ± s.e.m.; n = 3–4 biological replicates).
Extended Data Figure 4 Efficient AAV production using cell supernatant.
a, Lentiviral and AAV vectors carrying GFP were used to test transduction efficiency. b, Primary cortical neurons were transduced with 300 and 250 µl supernatant derived from the same number of lentivirus- or AAV-transduced 293FT cells. Representative images of GFP expression were collected at 7 days post infection. Scale bars, 50 μm. c, The depicted process was developed for the production of AAV supernatant and subsequent transduction of primary neurons. 293FT cells were transfected with an AAV vector carrying the gene of interest, the AAV1 serotype packaging vector (pAAV1), and helper plasmid (pDF6) using PEI. 48 h later, the supernatant was collected and filtered through a 0.45 μm PVDF membrane. Primary neurons were then transduced with supernatant and remaining aliquots were stored at −80 °C. Stable levels of AAV construct expression were reached after 5–6 days. AAV supernatant production following this process can be used for production of up to 96 different viral constructs in 96-well format (used for TALE screen in neurons shown in Fig. 2c).
Extended Data Figure 5 Characterizing LITEs in neurons and in vivo.
a, Impact of light duty cycle on primary neuron health. The effect of light stimulation on primary cortical neuron health was compared for duty cycles of 7%, 0.8%, and no light conditions. Calcein was used to evaluate neuron viability. Bright-field images show cell morphology and integrity. Primary cortical neurons were stimulated with the indicated duty cycle for 24 h with 5 mW cm−2 of 466 nm light. Representative images, scale bar, 50 μm. Pulses were performed in the following manner: 7% duty cycle = 1 s pulse at 0.067 Hz, 0.8% duty cycle = 0.5 s pulse at 0.0167 Hz. b, Co-transduction efficiency of LITE components by AAV1/2 in vivo in mouse infralimbic cortex. Cells transduced by TALE(Grm2)–CIB1 alone, CRY2PHR–VP64 alone, or co-transduced were calculated as a percentage of all transduced cells (mean ± s.e.m.; n = 9 fields from 3 animals). c, Grm2 mRNA levels were determined in primary neurons transfected with individual LITE components. Primary neurons expressing TALE(Grm2)–CIB1 alone led to a similar increase in Grm2 mRNA levels as unstimulated cells expressing the complete LITE system (mean ± s.e.m.; n = 3–4 biological replicates).
Extended Data Figure 6 Effects of LITE component engineering on activation, background signal and fold induction.
Protein modifications were used to find LITE components resulting in reduced background transcriptional activation while improving induction ratio by light. In brief, nuclear localization signals and mutations in an endogenous nuclear export signal were used to improve nuclear import of the CRY2PHR–VP64 component. Several variations of CIB1 intended to either reduce nuclear localization or CIB1 transcriptional activation were pursued to reduce the contribution of the TALE–CIB1 component to background activity. The results of all tested combinations of CRY2PHR–VP64 and TALE–CIB1 are shown above. The table to the left of the bar graphs indicates the particular combination of domains/mutations used for each condition. Each row of the table and bar graphs contains the component details, light/no light activity, and induction ratio by light for the particular CRY2PHR/CIB1 combination. Combinations that resulted in both decreased background and increased fold induction compared to LITE1.0 are highlighted in green in the table column marked ‘+’ (t-test P < 0.05). See Supplementary Discussion for detailed explanation of each modification (mean ± s.e.m.; n = 2–3 biological replicates).
Extended Data Figure 7 Strategies for optimizing the LITE system.
a, In the absence of light, the TALE–CIB1 LITE component resides in the cytoplasm due to the absence of a nuclear localization signal, NLS (or the addition of a nuclear export signal, NES). The CRY2PHR–VP64 component containing a NLS on the other hand is actively imported into the nucleus on its own. b, In the presence of blue light, TALE–CIB1 binds to CRY2PHR. The NLS present in CRY2PHR–VP64 now mediates nuclear import of the complex of both LITE components, enabling them to activate transcription at the targeted locus. In addition to the LITE2.0 constructs, several CRY2PHR–VP64/TALE–CIB1 combinations from the engineered LITE component screen were of particular note. LITE1.9.0, which combined the α-importin NLS effector construct with a mutated endogenous NLS and Δ276–307 TALE–CIB1 construct, exhibited an induction ratio greater than 9 and an absolute light activation of more than 180. LITE1.9.1, which combined the unmodified CRY2PHR–VP64 with a mutated NLS, Δ318–334, AD5 NES TALE–CIB1 construct, achieved an induction ratio of 4 with a background activation of 1.06. A selection of other LITE1.9 combinations with background activations lower than 2 and induction ratios ranging from 7 to 12 were also highlighted (mean ± s.e.m.; n = 2–3 biological replicates).
Extended Data Figure 8 TALE SID4X repressor characterization and application in neurons.
a, A synthetic repressor was constructed by concatenating 4 SID domains (SID4X). To identify the optimal TALE-repressor architecture, SID or SID4X was fused to a TALE designed to target the mouse p11 (also known as S100a10) gene. b, Fold decrease in p11 mRNA was assayed using qRT–PCR (mean ± s.e.m.; n = 3 biological replicates). c, General schematic of constitutive TALE transcriptional repressor packaged into AAV. Effector domain SID4X is highlighted. hSyn, human synapsin promoter; 2A, Thosea asigna virus 2A self-cleaving peptide33; WPRE, woodchuck hepatitis post-transcriptional response element; bGH pA, bovine growth hormone poly-A signal. phiLOV2.134 (330 bp) was chosen as a shorter fluorescent marker to ensure efficient AAV packaging. d, A TALE targeting either the endogenous mouse locus Grm5 or Grm2 was fused to SID4X and virally transduced into primary neurons. SID4X-mediated target gene downregulation is shown for each TALE relative to levels in control neurons expressing GFP only (mean ± s.e.m.; n = 3–4 biological replicates).
Extended Data Figure 9 A diverse set of epiTALEs mediate transcriptional repression in neurons and Neuro 2a cells.
a, 24 different histone effector domains were each fused to a Grm2 targeting TALE. TALE–effector fusions were expressed in primary cortical mouse neurons using AAV transduction. Grm2 mRNA levels were measured using qRT–PCR relative to neurons transduced with GFP only. (*P < 0.05; mean ± s.e.m.; n = 2–3 biological replicates.) b, A total of 32 epiTALEs were transfected into Neuro2A cells. 20 of them mediated significant repression of the targeted Neurog2 locus (*P < 0.05; mean ± s.e.m.; n = 2–3 biological replicates).
Extended Data Figure 10 epiTALEs mediating transcriptional repression along with histone modifications in Neuro 2a cells.
a, TALEs fused to histone-deacetylating epigenetic effectors NcoR and SIRT3 targeting the murine Neurog2 locus in Neuro 2a cells were assayed for repressive activity on Neurog2 transcript levels (mean ± s.e.m.; n = 2–3 biological replicates). b, ChIP qRT–PCR showing a reduction in H3K9 acetylation at the Neurog2 promoter for NcoR and SIRT3 epiTALEs (mean ± s.e.m.; n = 2–3 biological replicates). c, The epigenetic effector PHF19 with known histone methyltransferase binding activity was fused to a TALE targeting Neurog2. Repression of Neurog2 mRNA levels was observed (mean ± s.e.m.; n = 2–3 biological replicates). d, ChIP qRT–PCR showing an increase in H3K27me3 levels at the Neurog2 promoter for the PHF19 epiTALE (mean ± s.e.m.; n = 2–3 biological replicates).
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Supplementary Information
This file contains Supplementary Methods, a Supplementary Discussion, Supplementary Tables 1–10, lists of the Supplementary Sequences and the Photostimulation Hardware Control Scripts and Supplementary references. (PDF 1005 kb)
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Konermann, S., Brigham, M., Trevino, A. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013). https://doi.org/10.1038/nature12466
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DOI: https://doi.org/10.1038/nature12466
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