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A CRISPR-Based Screen for Hedgehog Signaling Provides Insights Into Ciliary Function and Ciliopathies

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A CRISPR-Based Screen for Hedgehog Signaling Provides Insights Into Ciliary Function and Ciliopathies ARTICLES https://doi.org/10.1038/s41588-018-0054-7 A CRISPR-based screen for Hedgehog signaling provides insights into ciliary function and ciliopathies David K. Breslow 1,2,7*, Sascha Hoogendoorn 3,7, Adam R. Kopp2, David W. Morgens4, Brandon K. Vu2, Margaret C. Kennedy1, Kyuho Han4, Amy Li4, Gaelen T. Hess4, Michael C. Bassik4, James K. Chen 3,5* and Maxence V. Nachury 2,6* 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. he primary cilium is a surface-exposed microtubule-based approach. Indeed, most studies to date have searched for genes that compartment that serves as an organizing center for diverse either intrinsically affect cell growth or affect sensitivity to applied Tsignaling pathways1–3. Mutations affecting cilia cause ciliopa- perturbations16–23. thies, a group of developmental disorders including Joubert syn- Here, we engineered a Hh-pathway-sensitive reporter to enable drome, Meckel syndrome (MKS), nephronophthisis (NPHP), and an antibiotic-based selection platform. Combining this reporter Bardet–Biedl Syndrome. The defining symptoms of ciliopathies with a single guide RNA (sgRNA) lentiviral library targeting the include skeletal malformations, mental retardation, sensory defects, mouse genome, we conducted a CRISPR-based screen that system- obesity, and kidney cysts, and are thought to arise from misregu- atically identified ciliary components, Hh-signaling machinery, and lation of ciliary signaling pathways. Advances in human genetics ciliopathy genes with few false positives or false negatives. We fur- have led to the identification of more than 90 ciliopathy-associated ther show that previously uncharacterized hits encode new compo- genes4. However, the molecular basis for many ciliopathy cases nents of cilia and centrioles and also include novel ciliopathy genes. remains undiagnosed5, and critical aspects of cilium assembly and function remain poorly understood. Results A leading paradigm for ciliary signaling is the vertebrate Development of a Hh-pathway reporter for pooled screening. Hedgehog (Hh) pathway, which plays key roles in embryonic devel- Pooled functional screening requires the ability to enrich or deplete opment and in cancers such as medulloblastoma and basal cell mutants that exhibit a desired phenotype. Because ciliary signaling carcinoma6,7. Primary cilia are required for Hh-signaling output3, is not intrinsically linked to such a selectable phenotype, we engi- and all core components of the Hh-signaling machinery—from the neered a reporter that converts Hh signaling into antibiotic resis- receptor PTCH1 to the GLI transcriptional effectors—dynamically tance (Fig. 1a,b). This transcriptional reporter was introduced into localize to cilia during signal transduction. mouse NIH-3T3 fibroblasts, a cell line widely used to study Hh sig- Efforts to systematically identify genes needed for cilium assem- naling and cilium biology24, and these cells were then modified to bly or Hh signaling have been reported, but those studies have express Cas9-BFP (and are denoted 3T3-[Shh-BlastR;Cas9] cells). relied on arrayed short interfering RNA (siRNA) libraries and hence To validate our reporter cell line, we virally introduced sgRNAs exhibit the high rates of false positives and false negatives charac- targeting regulators of the Hh pathway (Supplementary Table 1). teristic of such screens8–11. Recently, genome-wide screening using The transmembrane receptor SMO and intraflagellar transport (IFT) CRISPR–Cas9 for gene disruption has emerged as a powerful tool complex subunit IFT88 are required for Hh signaling, whereas SUFU for functional genomics12–15. However, the pooled screening format and GLI3 restrain Hh-pathway activity (Fig. 1c, left). As expected, used in those studies requires a means to select for/against, or oth- sgRNAs targeting Smo or Ift88 severely decreased Sonic Hedgehog erwise isolate, cells exhibiting the desired phenotype, a requirement N-terminal domain (ShhN)-induced blasticidin resistance, whereas that has limited the scope of biological applications amenable to this deleting Gli3 potentiated blasticidin resistance, and targeting Sufu led 1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA. 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA. 3Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA. 4Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA. 5Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA. 6Present address: Department of Ophthalmology, UCSF, San Francisco, CA, USA. 7These authors contributed equally: David K. Breslow and Sascha Hoogendoorn. *e-mail: [email protected]; [email protected]; [email protected] 460 NatURE GENETICS | VOL 50 | MARCH 2018 | 460–471 | www.nature.com/naturegenetics © 2018 Nature America Inc., part of Springer Nature. All rights reserved. NATURE GENETICS ARTICLES a b –ShhN 100 +ShhN 50 8× Gli-BS Pmin BlastR Relative viability (%) 0 c SHH 0.11.0 10.0 DMSO Blasticidin (μg/ml) Positive regulators PTCH1 Negative regulators 150 ) –ShhN SMO 100 +ShhN g/ml μ ( 50 PKA 50 15 SUFU GLI2/3R 10 GLI2/3FL GLI2/3A 5 Blasticidin IC GLI1 Target genes 0 sgRNA: – Ctrl Sufu Gli3 Ift88 Smo-1 d sgRNA library (10 sgRNAs/gene) Gene A (positive Hh-pathway regulator) sgRNAs Gene B (negative Hh-pathway regulator) sgRNAs Gene C (noncilia/Hh-pathway gene) sgRNAs Control sgRNAs sgRNA lentivirus Two rounds signaling & blasticidin selection Selected Unselected Unselected 3T3-[Shh-BlastR;Cas9] cells sgRNA-infected cells Quantify sgRNAs by deep sequencing e Unselected +blasticidin f Other sgRNAs Other sgRNAs 125 1.00 100 79.8 Smo sgRNA-2 99.5 Smo sgRNA-2 20.2 100 0.48 sgRNA-2 80 o 75 Sm 60 0.10 40 50 –blasticidin Relative frequency 20 25 Relative % +blasticidin 0.01 0 0 0 1 2 101 102 103 104 105 101 102 103 104 105 Rounds of signaling GFP GFP & selection Fig. 1 | Development of a Hedgehog-pathway reporter-based screening strategy. a, A transcriptional reporter combining eight copies of the GLI binding sequence (8× Gli-BS) with a minimal promoter (Pmin) to convert Hh signals into blasticidin resistance. b, Blasticidin resistance was assayed across a range of concentrations in stimulated (+ ShhN) and unstimulated (–ShhN) 3T3-[Shh-BlastR;Cas9] cells. DMSO, dimethylsulfoxide control. Representative curves of 5 independent experiments performed in duplicate. c, Overview of the Hh pathway, with key negative and positive regulators shown in red and green, respectively (left). Effects of control sgRNAs on blasticidin resistance in stimulated and unstimulated 3T3-[Shh-BlastR;Cas9] cells (right). Bars show mean half-maximal inhibitory concentration (IC50) values, and circles show IC50 values from n = 2 (for gene-targeting sgRNAs) or 5 (for no sgRNA and negative- control (Ctrl) sgRNA) independent experiments performed in duplicate. d, Overview of the screening strategy. Cells receiving a negative-control sgRNA, a positive-regulator-targeting sgRNA, and a negative-regulator-targeting sgRNA are gray, green, and red, respectively. e, Flow cytometry histograms of cell mixtures showing the fraction of GFP-positive (Smo sgRNA-2, green) cells relative to cells expressing other library sgRNAs (blue) either in the absence of selection (left) or after two rounds of signaling and selection (right). Representative results from three independent experiments. f, Quantification of cell depletion in e. to ligand-independent blasticidin resistance (Fig. 1c, right). These We next tested the suitability of our reporter cells for pooled effects on blasticidin resistance were paralleled by concordant changes screening, which involves quantifying sgRNAs in blasticidin- in endogenous-pathway outputs, including GLI1 expression and selected and unselected cell pools to identify sgRNAs that con- changes in GLI3 processing (Supplementary Fig. 1a). Additionally, fer a selective advantage or disadvantage (Fig. 1d). We mimicked western blotting confirmed a loss of target-protein expression for screening conditions by mixing GFP-marked cells expressing a Gli3, Ift88, and Sufu sgRNAs (Supplementary Fig. 1a,b). Smo sgRNA with mCherry-marked cells expressing a portion of NatURE GENETICS | VOL 50 | MARCH 2018 | 460–471 | www.nature.com/naturegenetics 461 © 2018 Nature America Inc., part of Springer Nature. All rights reserved. ARTICLES NATURE GENETICS our genome-wide sgRNA library. Flow cytometry showed that the P = 9.6 × 10−61, and the Smoothened
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