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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Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331–344 (2010).
Nachury, M. V. How do cilia organize signalling cascades? Phil. Trans. R. Soc. Lond. B 369, 20130465 (2014).
Bangs, F. & Anderson, K. V. Primary cilia and mammalian Hedgehog signaling. Cold Spring Harb. Perspect. Biol. 9, a028175 (2017).
Braun, D. A. & Hildebrandt, F. Ciliopathies. Cold Spring Harb. Perspect. Biol. 9, a028191 (2017).
Akizu, N. et al. Mutations in CSPP1 lead to classical Joubert syndrome. Am. J. Hum. Genet. 94, 80–86 (2014).
Wu, F., Zhang, Y., Sun, B., McMahon, A. P. & Wang, Y. Hedgehog signaling: from basic biology to cancer therapy. Cell Chem. Biol. 24, 252–280 (2017).
Pak, E. & Segal, R. A. Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy. Dev. Cell 38, 333–344 (2016).
Roosing, S. et al. Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome. eLife 4, e06602 (2015).
Wheway, G. et al. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nat. Cell Biol. 17, 1074–1087 (2015).
Jacob, L. S. et al. Genome-wide RNAi screen reveals disease-associated genes that are common to Hedgehog and Wnt signaling. Sci. Signal. 4, ra4 (2011).
Kim, J. et al. Functional genomic screen for modulators of ciliogenesis and cilium length. Nature 464, 1048–1051 (2010).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).
Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).
Deans, R. M. et al. Parallel shRNA and CRISPR–Cas9 screens enable antiviral drug target identification. Nat. Chem. Biol. 12, 361–366 (2016).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Wang, T. et al. Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell 168, 890–903.e15 (2017).
Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163, 1515–1526 (2015).
Tzelepis, K. et al. A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep. 17, 1193–1205 (2016).
Orchard, R. C. et al. Discovery of a proteinaceous cellular receptor for a norovirus. Science 353, 933–936 (2016).
Park, R. J. et al. A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors. Nat. Genet. 49, 193–203 (2017).
Arroyo, J. D. et al. A genome-wide CRISPR death screen identifies genes essential for oxidative phosphorylation. Cell Metab. 24, 875–885 (2016).
Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).
Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nat. Commun. 8, 15178 (2017).
Morgens, D. W., Deans, R. M., Li, A. & Bassik, M. C. Systematic comparison of CRISPR/Cas9 and RNAi screens for essential genes. Nat. Biotechnol. 34, 634–636 (2016).
Hart, T., Brown, K. R., Sircoulomb, F., Rottapel, R. & Moffat, J. Measuring error rates in genomic perturbation screens: gold standards for human functional genomics. Mol. Syst. Biol. 10, 733 (2014).
Huang, W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Webb, T. R. et al. Diphthamide modification of eEF2 requires a J-domain protein and is essential for normal development. J. Cell Sci. 121, 3140–3145 (2008).
Chen, C. M. & Behringer, R. R. Ovca1 regulates cell proliferation, embryonic development, and tumorigenesis. Genes Dev. 18, 320–332 (2004).
Loucks, C. M. et al. Matching two independent cohorts validates DPH1 as a gene responsible for autosomal recessive intellectual disability with short stature, craniofacial, and ectodermal anomalies. Hum. Mutat. 36, 1015–1019 (2015).
Allen, B. L. et al. Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev. Cell 20, 775–787 (2011).
Izzi, L. et al. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev. Cell 20, 788–801 (2011).
Brinkman, E. K., Chen, T., Amendola, M. & van Steensel, B. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Lee, Y. L. et al. Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function. Mol. Biol. Cell 25, 2919–2933 (2014).
Burke, M. C. et al. Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation. J. Cell Biol. 207, 123–137 (2014).
Glazer, A. M. et al. The Zn finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis. Dev. Biol. 337, 148–156 (2010).
Li, F. Q. et al. BAR domain-containing FAM92 proteins interact with Chibby1 to facilitate ciliogenesis. Mol. Cell. Biol. 36, 2668–2680 (2016).
Dorn, K. V., Hughes, C. E. & Rohatgi, R. A Smoothened-Evc2 complex transduces the Hedgehog signal at primary cilia. Dev. Cell 23, 823–835 (2012).
Pusapati, G. V. et al. EFCAB7 and IQCE regulate hedgehog signaling by tethering the EVC-EVC2 complex to the base of primary cilia. Dev. Cell 28, 483–496 (2014).
Ruiz-Perez, V. L. & Goodship, J. A. Ellis-van Creveld syndrome and Weyers acrodental dysostosis are caused by cilia-mediated diminished response to hedgehog ligands. Am. J. Med. Genet. C. Semin. Med. Genet. 151C, 341–351 (2009).
Shaheen, R. et al. Genomic analysis of Meckel-Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes. Eur. J. Hum. Genet. 21, 762–768 (2013).
Shaheen, R. et al. Characterizing the morbid genome of ciliopathies. Genome Biol. 17, 242 (2016).
Kar, A., Phadke, S. R., Das Bhowmik, A. & Dalal, A. Whole exome sequencing reveals a mutation in ARMC9 as a cause of mental retardation, ptosis and polydactyly. Preprint at https://www.biorxiv.org/content/early/2017/02/17/109124 (2017).
Panigrahi, I., Phadke, S. R. & Agarwal, S. S. Mental retardation, ptosis and polydactyly: a new autosomal recessive syndrome? Clin. Dysmorphol. 11, 289–292 (2002).
Nager, A. R. et al. An actin network dispatches ciliary GPCRs into extracellular vesicles to modulate signaling. Cell 168, 252–263.e14 (2017).
Phua, S. C. et al. Dynamic remodeling of membrane composition drives cell cycle through primary cilia excision. Cell 168, 264–279.e15 (2017).
Van De Weghe, J. C. et al. Mutations in ARMC9, which encodes a basal body protein, cause Joubert syndrome in humans and ciliopathy phenotypes in zebrafish. Am. J. Hum. Genet. 101, 23–36 (2017).
Xu, M. et al. Mutations in the spliceosome component CWC27 cause retinal degeneration with or without additional developmental anomalies. Am. J. Hum. Genet. 100, 592–604 (2017).
Sifrim, A. et al. Distinct genetic architectures for syndromic and nonsyndromic congenital heart defects identified by exome sequencing. Nat. Genet. 48, 1060–1065 (2016).
Oegema, R. et al. Human mutations in integrator complex subunits link transcriptome integrity to brain development. PLoS Genet. 13, e1006809 (2017).
Sollis, E. et al. Identification and functional characterization of de novo FOXP1 variants provides novel insights into the etiology of neurodevelopmental disorder. Hum. Mol. Genet. 25, 546–557 (2016).
Comartin, D. et al. CEP120 and SPICE1 cooperate with CPAP in centriole elongation. Curr. Biol. 23, 1360–1366 (2013).
Lin, Y. N. et al. CEP120 interacts with CPAP and positively regulates centriole elongation. J. Cell Biol. 202, 211–219 (2013).
Mahjoub, M. R., Xie, Z. & Stearns, T. Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly. J. Cell Biol. 191, 331–346 (2010).
Kanie, T. et al. The CEP19-RABL2 GTPase complex binds IFT-B to initiate intraflagellar transport at the ciliary base. Dev. Cell 42, 22–36.e12 (2017).
Mojarad, B. A. et al. CEP19 cooperates with FOP and CEP350 to drive early steps in the ciliogenesis programme. Open Biol. 7, 170114 (2017).
Nishijima, Y. et al. RABL2 interacts with the intraflagellar transport-B complex and CEP19 and participates in ciliary assembly. Mol. Biol. Cell 28, 1652–1666 (2017).
Gupta, G. D. et al. A dynamic protein interaction landscape of the human centrosome-cilium interface. Cell 163, 1484–1499 (2015).
Jakobsen, L. et al. Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods. EMBO J. 30, 1520–1535 (2011).
Knorz, V. J. et al. Centriolar association of ALMS1 and likely centrosomal functions of the ALMS motif-containing proteins C10orf90 and KIAA1731. Mol. Biol. Cell 21, 3617–3629 (2010).
Izquierdo, D., Wang, W. J., Uryu, K. & Tsou, M. F. Stabilization of cartwheel-less centrioles for duplication requires CEP295-mediated centriole-to-centrosome conversion. Cell Rep. 8, 957–965 (2014).
Chang, P. & Stearns, T. Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function. Nat. Cell Biol. 2, 30–35 (2000).
Dupuis-Williams, P. et al. Functional role of epsilon-tubulin in the assembly of the centriolar microtubule scaffold. J. Cell Biol. 158, 1183–1193 (2002).
Dutcher, S. K., Morrissette, N. S., Preble, A. M., Rackley, C. & Stanga, J. Epsilon-tubulin is an essential component of the centriole. Mol. Biol. Cell 13, 3859–3869 (2002).
O’Toole, E. T., Giddings, T. H., McIntosh, J. R. & Dutcher, S. K. Three-dimensional organization of basal bodies from wild-type and delta-tubulin deletion strains of Chlamydomonas reinhardtii. Mol. Biol. Cell 14, 2999–3012 (2003).
Ross, I., Clarissa, C., Giddings, T. H. Jr. & Winey, M. ε-tubulin is essential in Tetrahymena thermophila for the assembly and stability of basal bodies. J. Cell Sci. 126, 3441–3451 (2013).
Sharma, A. et al. Centriolar CPAP/SAS-4 imparts slow processive microtubule growth. Dev. Cell 37, 362–376 (2016).
Schmidt, T. I. et al. Control of centriole length by CPAP and CP110. Curr. Biol. 19, 1005–1011 (2009).
Kirkham, M., Müller-Reichert, T., Oegema, K., Grill, S. & Hyman, A. A. SAS-4 is a C. elegans centriolar protein that controls centrosome size. Cell 112, 575–587 (2003).
Hein, M. Y. et al. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163, 712–723 (2015).
Huttlin, E. L. et al. Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509 (2017).
Wong, Y. L. et al. Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science 348, 1155–1160 (2015).
Aguirre, A. J. et al. Genomic copy number dictates a gene-independent cell response to CRISPR/Cas9 targeting. Cancer Discov. 6, 914–929 (2016).
Wang, J. T., Kong, D., Hoerner, C. R., Loncarek, J. & Stearns, T. Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells. eLife 6, e29061 (2017).
Li, Y. et al. Global genetic analysis in mice unveils central role for cilia in congenital heart disease. Nature 521, 520–524 (2015).
MacArthur, D. G. et al. Guidelines for investigating causality of sequence variants in human disease. Nature 508, 469–476 (2014).
Pimenta-Marques, A. et al. A mechanism for the elimination of the female gamete centrosome in Drosophila melanogaster. Science 353, aaf4866 (2016).
Zheng, X. et al. Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length. Nat. Commun. 7, 11874 (2016).
Bond, J. et al. A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat. Genet. 37, 353–355 (2005).
Gilmore, E. C. & Walsh, C. A. Genetic causes of microcephaly and lessons for neuronal development. Wiley Interdiscip. Rev. Dev. Biol. 2, 461–478 (2013).
Liew, G. M. et al. The intraflagellar transport protein IFT27 promotes BBSome exit from cilia through the GTPase ARL6/BBS3. Dev. Cell 31, 265–278 (2014).
Frank-Kamenetsky, M. et al. Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists. J. Biol. 1, 10 (2002).
Chen, J. K., Taipale, J., Young, K. E., Maiti, T. & Beachy, P. A. Small molecule modulation of Smoothened activity. Proc. Natl. Acad. Sci. USA 99, 14071–14076 (2002).
Breslow, D. K., Koslover, E. F., Seydel, F., Spakowitz, A. J. & Nachury, M. V. An in vitro assay for entry into cilia reveals unique properties of the soluble diffusion barrier. J. Cell Biol. 203, 129–147 (2013).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Elias, J. E. & Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).
de Hoon, M. J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004).
Saldanha, A. J. Java Treeview: extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).
Turk, E. et al. Zeta-tubulin is a member of a conserved tubulin module and is a component of the centriolar basal foot in multiciliated cells. Curr. Biol. 25, 2177–2183 (2015).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Li, W. et al. The EMBL-EBI bioinformatics web and programmatic tools framework. Nucleic Acids Res. 43, W580–W584 (2015).
Perrière, G. & Gouy, M. WWW-query: an on-line retrieval system for biological sequence banks. Biochimie 78, 364–369 (1996).
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.
The authors declare no competing interests.
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Integrated supplementary information
. 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.
. 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.
. 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.
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).
. 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.
. 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).
. Tree diagrams showing sequence relationships for select proteins, with branch lengths scaled equally for all proteins. See also Supplementary Table 8.
. 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 Figures 1–8, Supplementary Note and Supplementary Tables 9 and 10
List of select sgRNA and primer sequences
List of sgRNA counts by deep sequencing of screen cell pools
casTLE output (gene scores) for genome-wide screen
Summary of screen data for select genes of interest
List of functional categories enriched among screen hits
List of proteins identified by mass spectrometry in affinity purifications
Clustered growth data from Aguirre et al
Evolutionary analysis of TED complex components
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Breslow, D.K., Hoogendoorn, S., Kopp, A.R. et al. A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies. Nat Genet 50, 460–471 (2018). https://doi.org/10.1038/s41588-018-0054-7
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