Abstract
Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. It is based on identifying cellular hypersensitivity and resistance caused by individual gene modulations with genome-wide coverage. Due to the requirement of bar-coded, genome-wide deletion collections, high-resolution experiments of this nature have historically been limited to fungal systems. Pooled RNAi reagents have enabled similar attempts in mammalian cells but efforts have been hampered by significant off-target effects and experimental noise. The CRISPR/Cas9 system for the first time enables precise DNA editing at defined loci in a genome-wide fashion. Here we present the detailed protocol that leverages the CRISPR/Cas9 system for chemogenomic profiling and target identification of diverse chemical probes.
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References
Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 231(25):232–235
Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, Michaud GA, Charlat O, Wiellette E, Zhang Y, Wiessner S, Hild M, Shi X, Wilson CJ, Mickanin C, Myer V, Fazal A, Tomlinson R, Serluca F, Shao W, Cheng H, Shultz M, Rau C, Schirle M, Schlegl J, Ghidelli S, Fawell S, Lu C, Curtis D, Kirschner MW, Lengauer C, Finan PM, Tallarico JA, Bouwmeester T, Porter JA, Bauer A, Cong F (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461(7264):614–620. https://doi.org/10.1038/nature08356
Heitman J, Movva NR, Hall MN (1991) Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253(5022):905–909
Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M'Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Veronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285(5429):901–906
Giaever G, Shoemaker DD, Jones TW, Liang H, Winzeler EA, Astromoff A, Davis RW (1999) Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat Genet 21(3):278–283. https://doi.org/10.1038/6791
Lee AY, St Onge RP, Proctor MJ, Wallace IM, Nile AH, Spagnuolo PA, Jitkova Y, Gronda M, Wu Y, Kim MK, Cheung-Ong K, Torres NP, Spear ED, Han MK, Schlecht U, Suresh S, Duby G, Heisler LE, Surendra A, Fung E, Urbanus ML, Gebbia M, Lissina E, Miranda M, Chiang JH, Aparicio AM, Zeghouf M, Davis RW, Cherfils J, Boutry M, Kaiser CA, Cummins CL, Trimble WS, Brown GW, Schimmer AD, Bankaitis VA, Nislow C, Bader GD, Giaever G (2014) Mapping the cellular response to small molecules using chemogenomic fitness signatures. Science 344(6180):208–211. https://doi.org/10.1126/science.1250217
Hoepfner D, Helliwell SB, Sadlish H, Schuierer S, Filipuzzi I, Brachat S, Bhullar B, Plikat U, Abraham Y, Altorfer M, Aust T, Baeriswyl L, Cerino R, Chang L, Estoppey D, Eichenberger J, Frederiksen M, Hartmann N, Hohendahl A, Knapp B, Krastel P, Melin N, Nigsch F, Oakeley EJ, Petitjean V, Petersen F, Riedl R, Schmitt EK, Staedtler F, Studer C, Tallarico JA, Wetzel S, Fishman MC, Porter JA, Movva NR (2014) High-resolution chemical dissection of a model eukaryote reveals targets, pathways and gene functions. Microbiol Res 169(2-3):107–120. https://doi.org/10.1016/j.micres.2013.11.004
Roemer T, Xu D, Singh SB, Parish CA, Harris G, Wang H, Davies JE, Bills GF (2011) Confronting the challenges of natural product-based antifungal discovery. Chem Biol 18(2):148–164. https://doi.org/10.1016/j.chembiol.2011.01.009
Whitehurst AW, Bodemann BO, Cardenas J, Ferguson D, Girard L, Peyton M, Minna JD, Michnoff C, Hao W, Roth MG, Xie XJ, White MA (2007) Synthetic lethal screen identification of chemosensitizer loci in cancer cells. Nature 446(7137):815–819. https://doi.org/10.1038/nature05697
Bassik MC, Kampmann M, Lebbink RJ, Wang S, Hein MY, Poser I, Weibezahn J, Horlbeck MA, Chen S, Mann M, Hyman AA, Leproust EM, McManus MT, Weissman JS (2013) A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152(4):909–922. https://doi.org/10.1016/j.cell.2013.01.030
Matheny CJ, Wei MC, Bassik MC, Donnelly AJ, Kampmann M, Iwasaki M, Piloto O, Solow-Cordero DE, Bouley DM, Rau R, Brown P, McManus MT, Weissman JS, Cleary ML (2013) Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens. Chem Biol 20(11):1352–1363. https://doi.org/10.1016/j.chembiol.2013.09.014
Estoppey D, Hewett JW, Guy CT, Harrington E, Thomas JR, Schirle M, Cuttat R, Waldt A, Gerrits B, Yang Z, Schuierer S, Pan X, Xie K, Carbone W, Knehr J, Lindeman A, Russ C, Frias E, Hoffman GR, Varadarajan M, Ramadan N, Reece-Hoyes JS, Wang Q, Chen X, McAllister G, Roma G, Bouwmeester T, Hoepfner D (2017) Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells. Sci Rep 7:42728. https://doi.org/10.1038/srep42728
Estoppey D, Lee CM, Janoschke M, Lee BH, Wan KF, Dong H, Mathys P, Filipuzzi I, Schuhmann T, Riedl R, Aust T, Galuba O, McAllister G, Russ C, Spiess M, Bouwmeester T, Bonamy GMC, Hoepfner D (2017) The natural product cavinafungin selectively interferes with Zika and dengue virus replication by inhibition of the host signal peptidase. Cell Rep 19(3):451–460. https://doi.org/10.1016/j.celrep.2017.03.071
Konig R, Chiang CY, Tu BP, Yan SF, DeJesus PD, Romero A, Bergauer T, Orth A, Krueger U, Zhou Y, Chanda SK (2007) A probability-based approach for the analysis of large-scale RNAi screens. Nat Methods 4(10):847–849. https://doi.org/10.1038/nmeth1089
Brodsky RA (2009) How I treat paroxysmal nocturnal hemoglobinuria. Blood 113(26):6522–6527. https://doi.org/10.1182/blood-2009-03-195966
Acknowledgments
We thank Nadire Ramadan Cochran, David Estoppey, Malini Varadarajan, and Claudia Agarinis for their help setting up the described protocols and careful proofreading of the manuscript.
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Hoepfner, D., McAllister, G., Hoffman, G.R. (2019). CRISPR/Cas9-Based Chemogenomic Profiling in Mammalian Cells. In: Ziegler, S., Waldmann, H. (eds) Systems Chemical Biology. Methods in Molecular Biology, vol 1888. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8891-4_9
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DOI: https://doi.org/10.1007/978-1-4939-8891-4_9
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