Primary cilia organize Hedgehog signaling and shape embryonic development, and their dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen can robustly identify factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovered novel components of several ciliary structures, including a protein complex that contains δ-tubulin and ε-tubulin and is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and showed that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies, and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

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We acknowledge members of the laboratories of J.K.C. and M.V.N. for advice and technical support; S. van Dorp for assistance with image quantification; P. Beachy (Stanford) for NIH-3T3 cells, 8×Gli-luciferase reporter plasmid, and ShhN-producing HEK293T cells; K. Anderson (Sloan Kettering) for antibody to SMO; G. Crabtree, K. C. Garcia, and A. Pyle for use of plate readers; N. Dimitrova for microscope use; C. Bustamante for use of an Illumina sequencer; R. Rohatgi (Stanford) for antibodies to EVC and IQCE and NIH-3T3 FlpIn cells; P. Jackson (Stanford) for IMCD3 FlpIn cells; J. Wang and T. Stearns (Stanford) for sharing unpublished results and cDNA for Cby1; and M. Scott for helpful discussions. This project was supported by NIH Pathway to Independence Award K99/R00 HD082280 (D.K.B.), Damon Runyon Dale F. Frey Award DFS-11-14 (D.K.B.), a seed grant from the Stanford Center for Systems Biology (D.K.B., S.H., and G.T.H.) and Stanford ChEM-H (M.C.B.), an NWO Rubicon Postdoctoral Fellowship (S.H.), National Science Foundation Graduate Research Fellowship DGE-114747 (D.W.M.), a Walter V. and Idun Berry Award (K.H.), and NIH T32 HG000044 (G.T.H.), DP2 HD084069 (M.C.B.), R01 GM113100 (J.K.C.), and R01 GM089933 (M.V.N.). Cell sorting/flow cytometry was done on instruments in the Stanford Shared FACS Facility, including an instrument supported by NIH shared instrument grant S10RR025518. Mass spectrometry analyses were conducted in the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University Mass Spectrometry and the Stanford Cancer Institute Proteomics/Mass Spectrometry Shared Resource; these centers are supported by Award S10 RR027425 from the National Center for Research Resources and NIH P30 CA124435, respectively. We thank C. Carstens, B. Borgo, P. Sheffield, and L. Bruhn of Agilent Technologies for cilia-focused oligonucleotide sublibraries.

Author information

Author notes

    • Maxence V. Nachury

    Present address: Department of Ophthalmology, UCSF, San Francisco, CA, USA

  1. These authors contributed equally: David K. Breslow and Sascha Hoogendoorn.


  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

    • David K. Breslow
    •  & Margaret C. Kennedy
  2. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    • David K. Breslow
    • , Adam R. Kopp
    • , Brandon K. Vu
    •  & Maxence V. Nachury
  3. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA

    • Sascha Hoogendoorn
    •  & James K. Chen
  4. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    • David W. Morgens
    • , Kyuho Han
    • , Amy Li
    • , Gaelen T. Hess
    •  & Michael C. Bassik
  5. Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA

    • James K. Chen


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D.K.B., S.H., J.K.C., and M.V.N. conceived the project with advice from M.C.B.; D.K.B. and S.H. developed the Hh-pathway reporter screening strategy with assistance from B.K.V.; and D.W.M., K.H., A.L., G.T.H., and M.C.B. provided functional genomics expertise, the genome-wide sgRNA library, and software for screen data analysis. D.K.B. conducted the genome-wide screen and screen data analysis with assistance from S.H. and A.R.K.; D.K.B., S.H., A.R.K., and M.C.K. functionally characterized hit genes of interest, analyzed data, and prepared figures. D.K.B., S.H., J.K.C., and M.V.N. wrote the manuscript with assistance from M.C.B.; D.K.B., S.H., G.T.H., M.C.B., J.K.C. and M.V.N. provided funding for the project.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to David K. Breslow or James K. Chen or Maxence V. Nachury.

Integrated supplementary information

  1. Supplementary Figure 1 Validation of positive- and negative-control sgRNAs

    . a) Lysates from cells transduced with the indicated sgRNAs were analyzed by western blot of the indicated proteins. GLI3-FL and GLI3-R indicate the positions of full-length and repressor forms of GLI3, respectively. Molecular weight marker is indicated by lane M. b) Lysates from cells transduced with the indicated sgRNAs were analyzed by western blot against IFT88 and loading control Importin β (Impβ). Representative blots are shown for one of two independent experiments. For full size blots, see Supplementary Figure 8.

  2. Supplementary Figure 2 Overview of sgRNA library and genome-wide-screen results

    . a) Description of sgRNA library used, indicating sub-libraries, number of sgRNAs per sub-library, and grouping of sub-libraries into four screen batches. b) Assessment of screen performance at detecting genes with growth phenotypes using 188 positive and 478 negative reference genes. Performance was determined by ROC curve (left) and precision-recall analysis (right), with the area under each curve (AUC) shown. Performance for various screens using different libraries are plotted, with the cell type and data source indicated for each. Dashed lines indicate performance of a random classification model. c) Relationship between growth and signaling phenotypes for genome-wide screen data. P-values were calculated using casTLE using 100,000 sgRNA permutations. See also Supplementary Table 3. Positive values indicate enrichment (increased growth or blasticidin resistance); negative values indicate depletion (reduced growth or blasticidin sensitivity). Genes shown in grey were filtered out of further analyses due to strong negative growth phenotypes.

  3. Supplementary Figure 3 Evaluation of screen performance

    . a) Comparison of hit gene detection rates across various screen datasets and functional classes of genes. The number of genes in each class is indicated above each group of bars. See Supplementary Table 4 for additional details. b) Comparison of screen replicates for 95 genes measured in two different screen batches. Note: a small amount of jitter was added during plotting so that overlapping data points can be resolved. c) Comparison of screen replicates for 263 genes analyzed for roles in response to ShhN-induced versus SAG-induced Hh signaling. Gas1 is highlighted in red as a prominent exception to the high level of concordance between screen results. P-values plotted in panels (b) and (c) were calculated using casTLE; see Methods and Supplementary Table 3.

  4. Supplementary Figure 4 Characterization of FAM92A and TTC23 as transition-zone and EvC-zone components

    . a) Analysis of Cas9-induced mutations in cell pools transduced with the indicated sgRNAs. Mutation frequencies were identified by TIDE sequencing (Tracking of Indels by Decomposition)34. Note that the sum of mutations for each gene is less than 100% because the TIDE algorithm only reports indels of up to 50 bp that can be identified with high confidence (P < 0.001). b) Signaling-induced blasticidin resistance is shown for 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs. Bars show mean IC50 values from N=5 (Ctrl; same data as shown in Fig. 1c), N=4 (Fam92a sgRNA) or N=3 (Ttc23 sgRNA) independent experiments (indicated by circles). c) Transfected CBY1-GFP co-localizes with endogenous FAM92A at the transition zone of IMCD3 cells. Line plot at right shows relative intensity from base to tip. Scale bar: 1 μm. Representative image from five fields of view. d) Validation of the anti-FAM92A antibody. Staining reveals prominent transition zone staining in wildtype (WT) cells but not cells transduced with a Fam92a-targeting sgRNA. Scale bar: 5 μm. Representative image of three independent experiments (five fields of view in each). e) TTC23-LAP stably expressed in IMCD3 cells co-localizes with EVC. Line scans at right show relative intensity from base to tip. Scale bar: 1 μm. Representative image of three independent experiments (five fields of view in each). f) The frequency of ciliogenesis was assessed in 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs. Bars show mean percentage of ciliated cells; dots show ciliated fraction in each independent experiment (at least 200 cells were analyzed in each of two independent experiments). g) Top: ciliary EVC fluorescence was measured for cells transduced with the indicated sgRNAs. The mean and standard error of the mean are plotted for N= 564, 277 and 285 cells expressing control (Ctrl), Ttc23-1 and Ttc23-2 sgRNAs, respectively. Data shown are from one of two representative experiments. Bottom: representative images showing EVC immunostaining in the indicated cells. Scale bars: 1 μm. h) Representative images showing ciliary IQCE staining in cells transduced with Ctrl and Ttc23-targeting sgRNAs. Scale bars: 5 μm. Representative images are shown from one of two independent experiments (five fields of view each).

  5. Supplementary Figure 5 Functional characterization of TXNDC15 and ARMC9

    . a) Distribution of cilium lengths measured for 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs. Control (Ctrl) sgRNA cells were significantly different from cells with Txndc15-1 sgRNA (P=4.4x10−10) or with Txndc15-2 sgRNA (P=3.3x10−10). Significance determined by two-sided Kolmogorov-Smirnov test; N = 902, 899, 1048 and 1242 cilia for WT, Ctrl, Txndc15-1 and Txndc15-2 sgRNAs, respectively. Data shown are drawn from 3 independent experiments. b) Staining for ciliary markers ARL13B and acetylated tubulin (AcTub) was performed for cells transduced with the indicated sgRNAs. Integrated ciliary fluorescence is shown relative to that of Ctrl sgRNA. Lines show means of N=3 independent experiments (symbols; at least 200 cilia analyzed per condition). c) Staining for ciliary markers ARL13B, acetylated tubulin (AcTub), and polyglutamylated tubulin (polyGlu-tub) was performed for 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs. Ciliary pixel intensity is shown relative to Ctrl sgRNA = 1. Lines show the mean of N=3 independent experiments (symbols; at least 200 cilia analyzed per condition). d) ARMC9-3xFLAG localization was assessed in NIH-3T3 FlpIn cells. Scale bar = 5 μm (1 μm for inset). Image shown is representative of three independent experiments (five fields of view in each). e) Plots of ARMC9-3xFLAG intensity along the length of the cilium from base (position 0) to tip (position 1.0) are shown for IMCD3 cells treated with ShhN in the presence or absence of Smo inhibitor vismodegib. The mean and standard deviation are plotted after normalizing the total intensity in each cilium to 1.0. f) Fluorescence intensity of GLI2 at the ciliary tip was measured for 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs and treated with ShhN or left untreated. Mean intensity (relative to Ctrl sgRNA cells +ShhN) and standard error of the mean are shown for each of N=3 experiments (at least 250 cilia analyzed for each condition). g) Fluorescence intensity of ciliary SMO was measured for cells transduced with the indicated sgRNAs and treated with ShhN or left untreated. Mean and 95% confidence interval are shown (N = number of cilia analyzed, one representative replicate out of two independent experiments). h) Representative micrographs for ciliary GLI2, GLI3 (three independent experiments), and SMO (two independent experiments) are shown for the indicated cell lines and treatment conditions. Scale bars = 1 μm.

  6. Supplementary Figure 6 Functional characterization of TEDC1 and TEDC2

    . a) Proteins affinity purified from IMCD3 wildtype (WT) or TEDC2-LAP cells were analyzed by western blotting with the indicated antibodies. See also Supplementary Figure 8. b) For blots shown in Fig. 6c, non-specific bands in anti-FLAG blot (marked by asterisks) show comparable protein loading among samples. Result is representative of two independent experiments. c) Signaling-induced blasticidin resistance is shown for 3T3-[Shh-BlastR;Cas9] cells transduced with the indicated sgRNAs. Bars show mean IC50 values from N=5 (Ctrl; same data as shown in Fig. 1c), N=3 (Tedc1 sgRNAs) or N=2 (Tedc2 sgRNAs) independent experiments (circles). d) Signaling-induced regulation of GLI3 processing and expression of target gene GLI1 were analyzed by Western blot using cells transduced with the indicated sgRNAs. GLI3-FL and GLI3-R indicate the positions of full-length and repressor forms of GLI3, respectively. Representative blot from two independent experiments is shown. See also Supplementary Figure 8. e) Centrioles marked by centrin2 and γ-tubulin were counted in cells transduced with the indicated sgRNAs. Following sgRNA introduction, TEDC1 rescue cells were transduced to express sgRNA-resistant TEDC1-3xFLAG-T2A-GFP (rescued cells are marked by GFP). N = 100, 149, 76 for Ctrl sgRNA, Tedc1-2 sgRNA, and TEDC1 rescue, respectively; * indicates P = 9.0x10−34; ** indicates P = 1.7x10−43; N.S. indicates not significant (P > 0.05), two-sided Fisher’s exact test. One of two representative experiments. f). Centrioles were stained in the indicated cells using antibodies to centrin2 and γ-tubulin. Cilia were stained using an antibody to ARL13B. Representative results from one of three independent experiments are shown. g) TEDC1-3xFLAG localizes to centrioles in rescued cells. Representative results from one of two independent experiments are shown. Scale bars: 5 μm (2 μm for inset). h) For cells transduced with the indicated sgRNAs, centrioles were visualized by staining with antibodies to centrin3 and γ-tubulin. Insets show centrin3 staining in mitotic cells, marked by yellow arrowheads. Scale bars: 5 μm (2 μm for insets). Images shown are from one of two independent experiments (10 fields of view each).

  7. Supplementary Figure 7 Evolutionary analysis of TED-complex components

    . Tree diagrams showing sequence relationships for select proteins, with branch lengths scaled equally for all proteins. See also Supplementary Table 8.

  8. Supplementary Figure 8 Full size western blots

    . Uncropped western blots for the indicated Figures. For Fig. 6c and Supplementary Fig. 6b, lanes 1-3 refer to cells transfected with: TEDC1-6xMYC and TagRFP-3xFLAG (lane 1); TagRFP-6xMYC and TEDC2-3xFLAG (lane 2); and TEDC1-6xMYC and TEDC2-3xFLAG (lane 3). In other panels, labels “-“, “+”, and “M” refer to unstimulated cells, ShhN-stimulated cells, and molecular weight marker lanes, respectively.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Note and Supplementary Tables 9 and 10

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    List of select sgRNA and primer sequences

  4. Supplementary Table 2

    List of sgRNA counts by deep sequencing of screen cell pools

  5. Supplementary Table 3

    casTLE output (gene scores) for genome-wide screen

  6. Supplementary Table 4

    Summary of screen data for select genes of interest

  7. Supplementary Table 5

    List of functional categories enriched among screen hits

  8. Supplementary Table 6

    List of proteins identified by mass spectrometry in affinity purifications

  9. Supplementary Table 7

    Clustered growth data from Aguirre et al

  10. Supplementary Table 8

    Evolutionary analysis of TED complex components

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