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Complex transcriptional modulation with orthogonal and inducible dCas9 regulators

Abstract

The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR–dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR–dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.

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Figure 1: A modular dCas9 platform for inducible gene activation and repression.
Figure 2: Characterization of the dynamics and dose response of ABA- and GA-inducible gene activation.
Figure 3: Orthogonal gene regulation by independently inducible dCas9s.
Figure 4: A multi-input CRISPR system for complex regulation of gene expression.
Figure 5: Transcriptome engineering using orthogonal and inducible dCas9 regulators.

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Acknowledgements

The authors thank the members of the Qi and Lim labs for advice and helpful discussions and the Stanford Shared FACS Facility for technical support. The authors also thank X. Shu (UCSF), F. Zhang (MIT), G. Crabtree (Stanford), T. Inoue (Johns Hopkins), T. Meyer (Stanford), C. Tucker (U of Colorado), and R. Dolmetsch (Stanford) for constructs used in this study. L.S.Q. acknowledges support from the NIH Office of the Director (OD) and the National Institute of Dental & Craniofacial Research (NIDCR). Y.G. acknowledges support from the Stanford Cancer Biology Graduate Program and the NSF GRFP fellowship. X.X. acknowledges the support from the Helen Hay Whitney Foundation postdoctoral fellowship. This work was supported by DP5 OD017887 (L.S.Q.), NIH R01 DA036858 (L.S.Q. and W.A.L.), NIH P50 GM081879 (W.A.L.), and the Howard Hughes Medical Institute (W.A.L.).

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Contributions

L.S.Q. and W.A.L. conceived of the research; Y.G. and X.X. designed the study; Y.G., X.X., S.W., and E.J.C. performed the experiments; Y.G., X.X., S.W., and L.S.Q. analyzed the data; Y.G., X.X., and L.S.Q. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Lei S Qi.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Screen of chemical- and light-mediated heterodimerization systems for inducible dCas9 gene activation.

(a) PiggyBac single vector design for expression of dCas9 and effector cassettes. (b) Binding sites of Sp sgTRE3G on the pTRE3G promoter. (c-f) Fluorescence quantification after 48 h induction for HEK293T pTRE3G-EGFP cells transfected with Sp TRE3G and (c) rapamycin-inducible, (d) PHYB/PIF red light-inducible, (e) CRY2PHR/CIBN blue light inducible, or (f) FKF1/GI blue light-inducible VPR-Sp dCas9. The data are displayed as mean ± SD for two (d-f) or four (c) independent transfections performed in technical replicates (n = 4 or 8). (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = not significant, Games-Howell post-hoc test following Welch’s ANOVA).

Supplementary Figure 2 Gene activation and repression by direct fusion dCas9s.

(a) Fluorescence quantification for HEK293T pTRE3G-EGFP cells 3 days following transfection with Sp sgTRE3G and VPR-Sp dCas9. (b) Fluorescence quantification for HEK293T pSV40-EGFP cells 6 days following transfection with Sp sgSV40 and KRAB-Sp dCas9. (c) Fluorescence quantification for HEK293T pTRE3G-EGFP cells 3 days following transfection with Sa sgTRE3G and VPR-Sa dCas9. Fold-change activation is indicated in black, while inverse fold-change repression is indicated in magenta. The data are displayed as mean ± SD for four independent transfections performed in technical replicates (n = 8). (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = not significant, Welch’s two-sided t test).

Supplementary Figure 3 Gene activation by different configurations of inducible dCas9s.

(a-c) Fluorescence quantification after 48 h induction for HEK293T pTRE3G-EGFP cells stably expressing Sp sgTRE3G and one of two configurations of (a) ABA-inducible, (b) PHYB/PIF red light-inducible, or (c) rapamycin-inducible VP64-Sp dCas9. The data are displayed as mean ± SD for two independent platings of a stable cell line performed in technical replicates (n = 4). Data collected from a separate experiment consisting of one plating. (d-f) Fluorescence quantification after 48 h induction for HEK293T pTRE3G-EGFP cells transfected with Sp sgTRE3G and one of two configurations of (d) GA-inducible, (e) CRY2PHR/CIBN blue light-inducible, or (f) FKF1/GI blue light-inducible VP64-Sp dCas9. The data are displayed as mean ± SD for two independent transfections performed in technical replicates (n = 4).

Supplementary Figure 4 Gene repression characterization for ABA-inducible KRAB-Sp dCas9.

(a) Binding sites of Sp sgSV40 on the pSV40 promoter. (b) 7 d timecourse for clonal HEK293T pSV40-EGFP cells stably expressing Sp sgSV40 and ABA-inducible KRAB-Sp dCas9. Cells were induced with ABA for 7 d (ON7) or for 4 and 5 d followed by ABA removal (ON4 OFF3 and ON5 OFF2). (c) ABA dose response after 5 d induction for clonal HEK293T pSV40-EGFP cells stably expressing Sp sgSV40 and ABA-inducible KRAB-Sp dCas9. The data are displayed as mean ± SD for two (b) or four (c) independent platings of a stable cell line (n = 2 or 4).

Supplementary Figure 5 Orthogonality of inducible dCas9 components.

(a) Fully orthogonal inducible dCas9 systems require each component of one system to avoid crosstalk to its counterpart in the paired system. (b) Fluorescence quantification after 48 h induction of HEK293T pTRE3G-EGFP cells transfected with Sp sgTRE3G and ABA-inducible or GA-inducible VP64-Sp dCas9. (c) Fluorescence quantification for HEK293T pTRE3G-EGFP cells 72 h following transfection with Sp/Sa sgTRE3G and direct fusion VPR-Sp/Sa dCas9. (d) Fluorescence quantification after 48 h induction of HEK293T pTRE3G-EGFP cells transfected with Sp/Sa sgTRE3G and ABA-inducible VP64-Sp/Sa dCas9. The data are displayed as mean ± SD for two independent transfections performed in technical replicates (n = 4). (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = not significant, Games-Howell post-hoc test following Welch’s ANOVA).

Supplementary Figure 6 Independent gene regulation by orthogonal dCas9s.

(a) 2-D flow cytometry contour plots of EGFP and mCherry fluorescence quantification after 5 d induction for experiment shown in Fig. 3b. The plots display data from one of four independent transfections for each condition. (b) 2-D flow cytometry contour plots of CD95 and CXCR4 immunofluorescence quantification after 48 h induction for experiment shown in Fig. 3d. The plots display data from one of four independent transfections for each condition.

Supplementary Figure 7 Two-input logic gates for inducible gene activation and repression.

(a) Fluorescence quantification after 48 h induction for HEK293T pTRE3G-EGFP cells transfected with Sp sgTRE3G and one of two configurations of an AND gate VP64-Sp dCas9. (b-c) Fluorescence quantifications after 5 d induction for HEK293T pSV40-EGFP cells transiently transfected with Sp sgSV40 and (a) the NOR gate or (b) the NAND gate KRAB-Sp dCas9 construct. Fold-change activation is indicated in black, while inverse fold-change repression is indicated in magenta. The data are displayed as mean ± SD for two (a, c) or four (b) independent transfections performed in technical replicates (n = 4 or 8). (* P < 0.05, ** P < 0.01, *** P < 0.001, n.s. = not significant, Games-Howell post-hoc test following Welch’s ANOVA).

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Gao, Y., Xiong, X., Wong, S. et al. Complex transcriptional modulation with orthogonal and inducible dCas9 regulators. Nat Methods 13, 1043–1049 (2016). https://doi.org/10.1038/nmeth.4042

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