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Enhancer RNAs pp 235-250 | Cite as

Targeted Gene Activation Using RNA-Guided Nucleases

  • Alexander Brown
  • Wendy S. Woods
  • Pablo Perez-PineraEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1468)

Abstract

The discovery of the prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system and its adaptation for targeted manipulation of DNA in diverse species has revolutionized the field of genome engineering. In particular, the fusion of catalytically inactive Cas9 to any number of transcriptional activator domains has resulted in an array of easily customizable synthetic transcription factors that are capable of achieving robust, specific, and tunable activation of target gene expression within a wide variety of tissues and cells. This chapter describes key experimental design considerations, methods for plasmid construction, gene delivery protocols, and procedures for analysis of targeted gene activation in mammalian cell lines using CRISPR-Cas transcription factors.

Key words

Genome engineering Synthetic biology RNA-guided nucleases CRISPR-Cas9 Gene expression Gene activation Transcription 

References

  1. 1.
    Uil TG, Haisma HJ, Rots MG (2003) Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. Nucleic Acids Res 31(21):6064–6078CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Knauert MP, Glazer PM (2001) Triplex forming oligonucleotides: sequence-specific tools for gene targeting. Hum Mol Genet 10(20):2243–2251CrossRefPubMedGoogle Scholar
  3. 3.
    Dervan PB, Edelson BS (2003) Recognition of the DNA minor groove by pyrrole-imidazole polyamides. Curr Opin Struct Biol 13(3):284–299CrossRefPubMedGoogle Scholar
  4. 4.
    Eguchi A, Lee GO, Wan F et al (2014) Controlling gene networks and cell fate with precision-targeted DNA-binding proteins and small-molecule-based genome readers. Biochem J 462(3):397–413CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Polstein LR, Perez-Pinera P, Kocak DD et al (2015) Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res 25(8):1158–1169CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Konermann S, Brigham MD, Trevino AE et al (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517(7536):583–588CrossRefPubMedGoogle Scholar
  7. 7.
    Perez-Pinera P, Kocak DD, Vockley CM et al (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Perez-Pinera P, Ousterout DG, Brunger JM et al (2013) Synergistic and tunable human gene activation by combinations of synthetic transcription factors. Nat Methods 10(3):239–242CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Maeder ML, Linder SJ, Reyon D et al (2013) Robust, synergistic regulation of human gene expression using TALE activators. Nat Methods 10(3):243–245CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Gilbert LA, Horlbeck MA, Adamson B et al (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159(3):647–661CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hilton IB, D'Ippolito AM, Vockley CM et al (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol 33(5):510–517CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chavez A, Scheiman J, Vora S et al (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12(4):326–328CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gersbach CA, Perez-Pinera P (2014) Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine. Expert Opin Ther Targets 18(8):835–839CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Gilbert LA, Larson MH, Morsut L et al (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Maeder ML, Linder SJ, Cascio VM et al (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10(10):977–979CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Cheng AW, Wang H, Yang H et al (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23(10):1163–1171CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Mali P, Aach J, Stranges PB et al (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31(9):833–838CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Farzadfard F, Perli SD, Lu TK (2013) Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth Biol 2(10):604–613CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096CrossRefPubMedGoogle Scholar
  20. 20.
    Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. doi: 10.1126/science CrossRefPubMedGoogle Scholar
  23. 23.
    Benchling (2015) Biology SoftwareGoogle Scholar
  24. 24.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Doench JG, Hartenian E, Graham DB et al (2014) Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol 32(12):1262–1267CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hsu PD, Scott DA, Weinstein JA et al (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Fu Y, Sander JD, Reyon D et al (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32(3):279–284CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Seipel K, Georgiev O, Schaffner W (1992) Different activation domains stimulate transcription from remote (“enhancer”) and proximal (“promoter”) positions. EMBO J 11(13):4961–4968PubMedPubMedCentralGoogle Scholar
  29. 29.
    Sadowski I, Ma J, Triezenberg S, Ptashne M (1988) GAL4-VP16 is an unusually potent transcriptional activator. Nature 335(6190):563–564CrossRefPubMedGoogle Scholar
  30. 30.
    Beerli RR, Segal DJ, Dreier B, Barbas CF 3rd (1998) Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. Proc Natl Acad Sci U S A 95(25):14628–14633CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Konermann S, Brigham MD, Trevino AE et al (2013) Optical control of mammalian endogenous transcription and epigenetic states. Nature 500(7463):472–476PubMedPubMedCentralGoogle Scholar
  32. 32.
    Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem Biol 11(3):198–200CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Polstein LR, Gersbach CA (2012) Light-inducible spatiotemporal control of gene activation by customizable zinc finger transcription factors. J Am Chem Soc 134(40):16480–16483CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Tanenbaum ME, Gilbert LA, Qi LS et al (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159(3):635–646CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Nissim L, Perli SD, Fridkin A et al (2014) Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Mol Cell 54(4):698–710CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Shechner DM, Hacisuleyman E, Younger ST, Rinn JL (2015) Multiplexable, locus-specific targeting of long RNAs with CRISPR-display. Nat Methods 12(7):664–670CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Kearns NA, Genga RM, Enuameh MS et al (2014) Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development 141(1):219–223CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Heckl D, Kowalczyk MS, Yudovich D et al (2014) Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat Biotechnol 32(9):941–946CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA (2014) Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res 42(19):e147CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Li HL, Fujimoto N, Sasakawa N et al (2015) Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9. Stem Cell Rep 4(1):143–154CrossRefGoogle Scholar
  41. 41.
    Shen B, Zhang W, Zhang J et al (2014) Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods 11(4):399–402CrossRefPubMedGoogle Scholar
  42. 42.
    Zheng Q, Cai X, Tan MH et al (2014) Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57(3):115–124CrossRefPubMedGoogle Scholar
  43. 43.
    Tsai SQ, Wyvekens N, Khayter C et al (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32(6):569–576CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Kleinstiver BP, Prew MS, Tsai SQ et al (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523(7561):481–485CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Ding Q, Regan SN, Xia Y et al (2013) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12(4):393–394CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Qi LS, Larson MH, Gilbert LA et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Wang T, Wei JJ, Sabatini DM, Lander ES (2014) Genetic screens in human cells using the CRISPR-Cas9 system. Science 343(6166):80–84CrossRefPubMedGoogle Scholar
  48. 48.
    Zhou Y, Zhu S, Cai C et al (2014) High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509(7501):487–491CrossRefPubMedGoogle Scholar
  49. 49.
    Koike-Yusa H, Li Y, Tan EP et al (2014) Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat Biotechnol 32(3):267–273CrossRefPubMedGoogle Scholar
  50. 50.
    Ranganathan V, Wahlin K, Maruotti J, Zack DJ (2014) Expansion of the CRISPR-Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs. Nat Commun 5:4516CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Platt RJ, Chen S, Zhou Y et al (2014) CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 159(2):440–455CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Swiech L, Heidenreich M, Banerjee A et al (2015) In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33(1):102–106CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Alexander Brown
    • 1
  • Wendy S. Woods
    • 1
  • Pablo Perez-Pinera
    • 1
    Email author
  1. 1.Department of BioengineeringUniversity of Illinois at Urbana-ChampaignUrbanaUSA

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