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Functional Genomics in Pharmaceutical Drug Discovery

Chapter
Part of the Handbook of Experimental Pharmacology book series (HEP, volume 232)

Abstract

Targeted therapies in personalized medicine require the knowledge about the molecular changes within the patient that cause the disease. With the beginning of the new century, a plethora of new technologies became available to detect these changes and use this information as starting point for drug development. Next-generation genome sequencing and sophisticated genome-wide functional genomics’ methods have led to a significant increase in the identification of novel drug target candidates and understanding of the relevance of these genomic and molecular changes for the diseases. As functional genomic tool for target identification, high-throughput gene silencing through RNA interference screening has become the established method. RNAi is discussed with its advantages and challenges in this chapter. Furthermore the potential of CRISPR/Cas9, a gene-editing method that has recently been adapted for use as functional screening tool, will be briefly reviewed.

Keywords

CRISPR/Cas9 Functional genomics High-content assay High-throughput screening RNA interference (RNAi) Short hairpin RNA (shRNA) Short interfering RNA (siRNA) 

Abbreviations

Cas9

CRISPR-associated nuclease

CCLE

Cancer cell line encyclopedia

CRISPR/Cas9

Clustered regularly interspaced short palindromic repeats/Cas9

CRISPRa

CRISPR activation

CRISPRi

CRISPR interference

dsRNA

Double-stranded DNA

DNA

Deoxyribonucleic acid

esiRNA

Endoribonuclease-prepared siRNA

FACS

Fluorescence-activated cell sorting

GoF

Gain of function

HCS

High-content screening

LoF

Loss of function

miRNA

MicroRNA

NGS

Next-generation sequencing

NHEJ

Nonhomologous end join

OTE

Off-target effect

RISC

RNA-induced silencing complex

RNAi

RNA interference

sgRNA

(Small) guide RNA

shRNA

Small hairpin RNA

siRNA

Small interfering RNA

UTR

Untranslated region

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Notes

Acknowledgments

We very much thank Anne Adams for her help with designing the figures.

References

  1. Ameres SL, Martinez J, Schroeder R (2007) Cell 130:101–112CrossRefPubMedGoogle Scholar
  2. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, Wilson CJ, Lehar J, Kryukov GV, Sonkin D, Reddy A, Liu M, Murray L, Berger MF, Monahan JE, Morais P, Meltzer J, Korejwa A, Jane-Valbuena J, Mapa FA, Thibault J, Bric-Furlong E, Raman P, Shipway A, Engels IH, Cheng J, Yu GK, Yu J, Aspesi P, de Silva M, Jagtap K, Jones MD, Wang L, Hatton C, Palescandolo E, Gupta S, Mahan S, Sougnez C, Onofrio RC, Liefeld T, MacConaill L, Winckler W, Reich M, Li N, Mesirov JP, Gabriel SB, Getz G, Ardlie K, Chan V, Myer VE, Weber BL, Porter J, Warmuth M, Finan P, Harris JL, Meyerson M, Golub TR, Morrissey MP, Sellers WR, Schlegel R, Garraway LA (2012) The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483:603–607CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bassik MC, Lebbink RJ, Churchman LS, Ingolia NT, Patena W, LeProust EM, Schuldiner M, Weissman JS, McManus MT (2009) Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat Methods 6:443–445CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bernards R, Brummelkamp TR, Beijersbergen RL (2006) shRNA libraries and their use in cancer genetics. Nature Methods 3:701–706CrossRefPubMedGoogle Scholar
  5. Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall WS, Khvorova A (2006) 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3:199–204CrossRefPubMedGoogle Scholar
  6. Birmingham A, Selfors LM, Forster T, Wrobel D, Kennedy CJ, Shanks E, Santoyo-Lopez J, Dunican DJ, Long A, Kelleher D, Smith Q, Beijersbergen RL, Ghazal P, Shamu CE (2009) Statistical methods for analysis of high-throughput RNA interference screens. Nat Methods 6:569–575CrossRefPubMedPubMedCentralGoogle Scholar
  7. Boutros M, Ahringer J (2008) The art and design of genetic screens: RNA interference. Nat Rev Genet 9:554–566CrossRefPubMedGoogle Scholar
  8. Brideau C, Gunter B, Pikounis B, Liaw A (2003) Improved statistical methods for hit selection in high-throughput screening. J Biomol Screen 8:634–647CrossRefPubMedGoogle Scholar
  9. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296:550–553CrossRefPubMedGoogle Scholar
  10. Buehler E, Chen YC, Martin S (2012a) C911: a bench-level control for sequence specific siRNA off-target effects. PLoS One 7:14CrossRefGoogle Scholar
  11. Buehler E, Khan AA, Marine S, Rajaram M, Bahl A, Burchard J, Ferrer M (2012b) siRNA off-target effects in genome-wide screens identify signaling pathway members. Sci Rep 2:428Google Scholar
  12. Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23:1163–1171CrossRefPubMedPubMedCentralGoogle Scholar
  13. Cheung HW, Cowley GS, Weir BA, Boehm JS, Rusin S, Scott JA, East A, Ali LD, Lizotte PH, Wong TC, Jiang G, Hsiao J, Mermel CH, Getz G, Barretina J, Gopal S, Tamayo P, Gould J, Tsherniak A, Stransky N, Luo B, Ren Y, Drapkin R, Bhatia SN, Mesirov JP, Garraway LA, Meyerson M, Lander ES, Root DE, Hahn WC (2011) Systematic investigation of genetic vulnerabilities across cancer cell lines reveals lineage-specific dependencies in ovarian cancer. Proc Natl Acad Sci U S A 108:12372–12377CrossRefPubMedPubMedCentralGoogle Scholar
  14. Diehl P, Tedesco D, Chenchik A (2014) Use of RNAi screens to uncover resistance mechanisms in cancer cells and identify synthetic lethal interactions. Drug Discov Today Technol 11:11–18CrossRefPubMedGoogle Scholar
  15. Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32:1262–1267CrossRefPubMedPubMedCentralGoogle Scholar
  16. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498CrossRefPubMedGoogle Scholar
  17. Fehrmann RS, Karjalainen JM, Krajewska M, Westra HJ, Maloney D, Simeonov A, Pers TH, Hirschhorn JN, Jansen RC, Schultes EA, van Haagen HH, de Vries EG, Te Meerman GJ, Wijmenga C, van Vugt MA, Franke L (2015) Gene expression analysis identifies global gene dosage sensitivity in cancer. Nat Genet 47:115–125CrossRefPubMedGoogle Scholar
  18. Fennell M, Xiang Q, Hwang A, Chen C, Huang C-H, Chen C-C, Pelossof R, Garippa RJ (2014) Impact of RNA-guided technologies for target identification and deconvolution. J Biomol Screen 19:1327–1337CrossRefPubMedGoogle Scholar
  19. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811CrossRefPubMedGoogle Scholar
  20. Gilbert Luke A, Horlbeck Max A, Adamson B, Villalta Jacqueline E, Chen Y, Whitehead Evan H, Guimaraes C, Panning B, Ploegh Hidde L, Bassik Michael C, Qi Lei S, Kampmann M, Weissman Jonathan S (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159:647–661CrossRefPubMedPubMedCentralGoogle Scholar
  21. Grimm S (2004) The art and design of genetic screens: mammalian culture cells. Nat Rev Genet 5:179–189CrossRefPubMedGoogle Scholar
  22. Guilinger JP, Thompson DB, Liu DR (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechol 32:577–582CrossRefGoogle Scholar
  23. Helming KC, Wang X, Wilson BG, Vazquez F, Haswell JR, Manchester HE, Kim Y, Kryukov GV, Ghandi M, Aguirre AJ, Jagani Z, Wang Z, Garraway LA, Hahn WC, Roberts CW (2014) ARID1B is a specific vulnerability in ARID1A-mutant cancers. Nat Med 20:251–254CrossRefPubMedPubMedCentralGoogle Scholar
  24. Hendel A, Fine EJ, Bao G, Porteus MH (2015) Quantifying on- and off-target genome editing. Trends Biotechnol 33:132–140CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jackson AL, Bartz SR, Schelter J, Kobayashi SV, Burchard J, Mao M, Li B, Cavet G, Linsley PS (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 21:635–637CrossRefPubMedGoogle Scholar
  26. Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. Elife 29:00471Google Scholar
  27. Khan AA, Betel D, Miller ML, Sander C, Leslie CS, Marks DS (2009) Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs. Nat Biotechnol 27:549–555CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826CrossRefPubMedPubMedCentralGoogle Scholar
  29. Martin SE, Caplen NJ (2007) Annu Rev Genomics Hum Genet 8:81–108CrossRefPubMedGoogle Scholar
  30. McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA (2002) Gene silencing using micro-RNA designed hairpins. RNA 8:842–850CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mohr SE, Smith JA, Shamu CE, Neumuller RA, Perrimon N (2014) RNAi screening comes of age: improved techniques and complementary approaches. Nat Rev Mol Cell Biol 15:591–600CrossRefPubMedPubMedCentralGoogle Scholar
  32. Moore JD (2015) The impact of CRISPR–Cas9 on target identification and validation. Drug Discov Today 20(4):450–457CrossRefPubMedGoogle Scholar
  33. Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible Co-suppression of homologous genes in trans. Plant Cell 2:279–289CrossRefPubMedPubMedCentralGoogle Scholar
  34. Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS (2002) Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958CrossRefPubMedPubMedCentralGoogle Scholar
  35. Patel AC (2013) Clinical relevance of target identity and biology: implications for drug discovery and development. J Biomol Screen 18:1164–1185CrossRefPubMedGoogle Scholar
  36. Pelz O, Gilsdorf M, Boutros M (2010) Web cellHTS2: a web-application for the analysis of high-throughput screening data. BMC Bioinformatics 11:1471–2105CrossRefGoogle Scholar
  37. Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32:347–355CrossRefPubMedPubMedCentralGoogle Scholar
  38. Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11:783–784CrossRefPubMedPubMedCentralGoogle Scholar
  39. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343:84–87CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sigoillot FD, King RW (2011) Vigilance and validation: keys to success in RNAi screening. ACS Chem Biol 6:47–60CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sigoillot FD, Lyman S, Huckins JF, Adamson B, Chung E, Quattrochi B, King RW (2012) A bioinformatics method identifies prominent off-targeted transcripts in RNAi screens. Nat Methods 9(4):363–366CrossRefPubMedPubMedCentralGoogle Scholar
  42. Surendranath V, Theis M, Habermann BH, Buchholz F (2013) Designing efficient and specific endoribonuclease-prepared siRNAs. Methods Mol Biol 942:193–204CrossRefPubMedGoogle Scholar
  43. Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 342:80–84CrossRefGoogle Scholar
  44. Weinstein IB (2002) Cancer. Addiction to oncogenes–the Achilles heel of cancer. Science 297:63–64CrossRefPubMedGoogle Scholar
  45. Wenzel C, Riefke B, Grundemann S, Krebs A, Christian S, Prinz F, Osterland M, Golfier S, Rase S, Ansari N, Esner M, Bickle M, Pampaloni F, Mattheyer C, Stelzer EH, Parczyk K, Prechtl S, Steigemann P (2014) 3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions. Exp Cell Res 323:131–143CrossRefPubMedGoogle Scholar
  46. Wilson BG, Helming KC, Wang X, Kim Y, Vazquez F, Jagani Z, Hahn WC, Roberts CW (2014) Residual complexes containing SMARCA2 (BRM) underlie the oncogenic drive of SMARCA4 (BRG1) mutation. Mol Cell Biol 34:1136–1144CrossRefPubMedPubMedCentralGoogle Scholar
  47. Zhou Y, Zhu S, Cai C, Yuan P, Li C, Huang Y, Wei W (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509:487–491CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Bayer Pharma AGBerlinGermany

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