Dissecting Tissue-Specific Super-Enhancers by Integrating Genome-Wide Analyses and CRISPR/Cas9 Genome Editing

Shlyueva D, Stampfel G, Stark A. Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet. 2014;15(4):272–86. https://doi.org/10.1038/nrg3682. Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, et al. High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008;132(2):311–22. https://doi.org/10.1016/j.cell.2007.12.014. Johnson DS, Mortazavi A, Myers RM, Wold B. Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007;316(5830):1497–502. https://doi.org/10.1126/science.1141319. Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007;4(8):651–7. https://doi.org/10.1038/nmeth1068. Ong CT, Corces VG. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat Rev Genet. 2011;12(4):283–93. https://doi.org/10.1038/nrg2957. Ong CT, Corces VG. Enhancers: emerging roles in cell fate specification. EMBO Rep. 2012;13(5):423–30. https://doi.org/10.1038/embor.2012.52. Morin R, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh T, et al. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. BioTechniques. 2008;45(1):81–94. https://doi.org/10.2144/000112900. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63. https://doi.org/10.1038/nrg2484. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435(7042):646–51. https://doi.org/10.1038/nature03556. Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007;25(7):778–85. https://doi.org/10.1038/nbt1319. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science. 2009;326(5959):1509–12. https://doi.org/10.1126/science.1178811. Moscou MJ, Bogdanove AJ. A simple cipher governs DNA recognition by TAL effectors. Science. 2009;326(5959):1501. https://doi.org/10.1126/science.1178817. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186(2):757–61. https://doi.org/10.1534/genetics.110.120717. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. A TALE nuclease architecture for efficient genome editing. Nat Biotechnol. 2011;29(2):143–8. https://doi.org/10.1038/nbt.1755. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60(2):174–82. https://doi.org/10.1007/s00239-004-0046-3. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005;151(Pt 3):653–63. https://doi.org/10.1099/mic.0.27437-0. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(Pt 8):2551–61. https://doi.org/10.1099/mic.0.28048-0. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012;482(7385):331–8. https://doi.org/10.1038/nature10886. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21. https://doi.org/10.1126/science.1225829. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. https://doi.org/10.1126/science.1231143. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. https://doi.org/10.1126/science.1232033. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910–8. https://doi.org/10.1016/j.cell.2013.04.025. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186–91. https://doi.org/10.1038/nature14299. Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell. 2013;153(2):307–19. https://doi.org/10.1016/j.cell.2013.03.035. Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-Andre V, Sigova AA, et al. Super-enhancers in the control of cell identity and disease. Cell. 2013;155(4):934–47. https://doi.org/10.1016/j.cell.2013.09.053. Yin JW, Wang G. The mediator complex: a master coordinator of transcription and cell lineage development. Development. 2014;141(5):977–87. https://doi.org/10.1242/dev.098392. Vahedi G, Kanno Y, Furumoto Y, Jiang K, Parker SC, Erdos MR, et al. Super-enhancers delineate disease-associated regulatory nodes in T cells. Nature. 2015;520(7548):558–62. https://doi.org/10.1038/nature14154. Siersbaek R, Rabiee A, Nielsen R, Sidoli S, Traynor S, Loft A, et al. Transcription factor cooperativity in early adipogenic hotspots and super-enhancers. Cell Rep. 2014;7(5):1443–55. https://doi.org/10.1016/j.celrep.2014.04.042. Adam RC, Yang H, Rockowitz S, Larsen SB, Nikolova M, Oristian DS, et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature. 2015;521(7552):366–70. https://doi.org/10.1038/nature14289. Huang J, Liu X, Li D, Shao Z, Cao H, Zhang Y, et al. Dynamic control of enhancer repertoires drives lineage and stage-specific transcription during hematopoiesis. Dev Cell. 2016;36(1):9–23. https://doi.org/10.1016/j.devcel.2015.12.014. Shin HY, Willi M, HyunYoo K, Zeng X, Wang C, Metser G, et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat Genet. 2016;48(8):904–11. https://doi.org/10.1038/ng.3606. Pittius CW, Sankaran L, Topper YJ, Hennighausen L. Comparison of the regulation of the whey acidic protein gene with that of a hybrid gene containing the whey acidic protein gene promoter in transgenic mice. Mol Endocrinol. 1988;2(11):1027–32. https://doi.org/10.1210/mend-2-11-1027. Robinson GW, Kang K, Yoo KH, Tang Y, Zhu BM, Yamaji D, et al. Coregulation of genetic programs by the transcription factors NFIB and STAT5. Mol Endocrinol. 2014;28(5):758–67. https://doi.org/10.1210/me.2012-1387. Zhou J, Chehab R, Tkalcevic J, Naylor MJ, Harris J, Wilson TJ, et al. Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation. EMBO J. 2005;24(3):635–44. https://doi.org/10.1038/sj.emboj.7600538. Lee HK, Willi M, Wang C, Yang CM, Smith HE, Liu C, et al. Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice. Nucleic Acids Res. 2017;45(8):4606–18. https://doi.org/10.1093/nar/gkx185. Willi M, Yoo KH, Reinisch F, Kuhns TM, Lee HK, Wang C, et al. Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells. Nat Commun. 2017;8:16069. https://doi.org/10.1038/ncomms16069. Mansour MR, Abraham BJ, Anders L, Berezovskaya A, Gutierrez A, Durbin AD, et al. Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science. 2014;346(6215):1373–7. https://doi.org/10.1126/science.1259037. Shi J, Whyte WA, Zepeda-Mendoza CJ, Milazzo JP, Shen C, Roe JS, et al. Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes Dev. 2013;27(24):2648–62. https://doi.org/10.1101/gad.232710.113. Drier Y, Cotton MJ, Williamson KE, Gillespie SM, Ryan RJ, Kluk MJ, et al. An oncogenic MYB feedback loop drives alternate cell fates in adenoid cystic carcinoma. Nat Genet. 2016;48(3):265–72. https://doi.org/10.1038/ng.3502. Parker SC, Stitzel ML, Taylor DL, Orozco JM, Erdos MR, Akiyama JA, et al. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. Proc Natl Acad Sci U S A. 2013;110(44):17921–6. https://doi.org/10.1073/pnas.1317023110. Achour M, Le Gras S, Keime C, Parmentier F, Lejeune FX, Boutillier AL, et al. Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice. Hum Mol Genet. 2015;24(12):3481–96. https://doi.org/10.1093/hmg/ddv099. Le Gras S, Keime C, Anthony A, Lotz C, De Longprez L, Brouillet E, et al. Altered enhancer transcription underlies Huntington's disease striatal transcriptional signature. Sci Rep. 2017;7:42875. https://doi.org/10.1038/srep42875. Shin HY. Targeting super-enhancers for disease treatment and diagnosis. Mol Cells. 2018;41(6):506–14. https://doi.org/10.14348/molcells.2018.2297. Zeng L, Zhou MM. Bromodomain: an acetyl-lysine binding domain. FEBS Lett. 2002;513(1):124–8. Zuber V, Bettella F, Witoelar A, Consortium P, Cruk G, Consortium B, et al. Bromodomain protein 4 discriminates tissue-specific super-enhancers containing disease-specific susceptibility loci in prostate and breast cancer. BMC Genomics. 2017;18(1):270. https://doi.org/10.1186/s12864-017-3620-y. Glodzik D, Morganella S, Davies H, Simpson PT, Li Y, Zou X, et al. A somatic-mutational process recurrently duplicates germline susceptibility loci and tissue-specific super-enhancers in breast cancers. Nat Genet. 2017;49(3):341–8. https://doi.org/10.1038/ng.3771. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell. 2013;153(2):320–34. https://doi.org/10.1016/j.cell.2013.03.036. Ohba S, He X, Hojo H, McMahon AP. Distinct transcriptional programs underlie Sox9 regulation of the mammalian chondrocyte. Cell Rep. 2015;12(2):229–43. https://doi.org/10.1016/j.celrep.2015.06.013. Liu CF, Lefebvre V. The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis. Nucleic Acids Res. 2015;43(17):8183–203. https://doi.org/10.1093/nar/gkv688. Wang AH, Juan AH, Ko KD, Tsai PF, Zare H, Dell’Orso S, et al. The elongation factor Spt6 maintains ESC pluripotency by controlling super-enhancers and counteracting Polycomb proteins. Mol Cell. 2017;68(2):398–413 e6. https://doi.org/10.1016/j.molcel.2017.09.016. Lomberk G, Blum Y, Nicolle R, Nair A, Gaonkar KS, Marisa L, et al. Distinct epigenetic landscapes underlie the pathobiology of pancreatic cancer subtypes. Nat Commun. 2018;9(1):1978. https://doi.org/10.1038/s41467-018-04383-6. Suzuki HI, Young RA, Sharp PA. Super-enhancer-mediated RNA processing revealed by integrative MicroRNA network analysis. Cell. 2017;168(6):1000–14 e15. https://doi.org/10.1016/j.cell.2017.02.015. Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J, et al. Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell. 2013;24(6):777–90. https://doi.org/10.1016/j.ccr.2013.11.003. Pelish HE, Liau BB, Nitulescu II, Tangpeerachaikul A, Poss ZC, Da Silva DH, et al. Mediator kinase inhibition further activates super-enhancer-associated genes in AML. Nature. 2015;526(7572):273–6. https://doi.org/10.1038/nature14904. Das S, Senapati P, Chen Z, Reddy MA, Ganguly R, Lanting L, et al. Regulation of angiotensin II actions by enhancers and super-enhancers in vascular smooth muscle cells. Nat Commun. 2017;8(1):1467. https://doi.org/10.1038/s41467-017-01629-7. Nakamura Y, Hattori N, Iida N, Yamashita S, Mori A, Kimura K, et al. Targeting of super-enhancers and mutant BRAF can suppress growth of BRAF-mutant colon cancer cells via repression of MAPK signaling pathway. Cancer Lett. 2017;402:100–9. https://doi.org/10.1016/j.canlet.2017.05.017. Gelato KA, Schockel L, Klingbeil O, Ruckert T, Lesche R, Toedling J, et al. Super-enhancers define a proliferative PGC-1alpha-expressing melanoma subgroup sensitive to BET inhibition. Oncogene. 2018;37(4):512–21. https://doi.org/10.1038/onc.2017.325. Andricovich J, Perkail S, Kai Y, Casasanta N, Peng W, Tzatsos A. Loss of KDM6A activates super-enhancers to induce gender-specific squamous-like pancreatic Cancer and confers sensitivity to BET inhibitors. Cancer Cell. 2018;33(3):512–26 e8. https://doi.org/10.1016/j.ccell.2018.02.003. Gerlach D, Tontsch-Grunt U, Baum A, Popow J, Scharn D, Hofmann MH, et al. The novel BET bromodomain inhibitor BI 894999 represses super-enhancer-associated transcription and synergizes with CDK9 inhibition in AML. Oncogene. 2018;37(20):2687–701. https://doi.org/10.1038/s41388-018-0150-2. Nakagawa M, Shaffer AL 3rd, Ceribelli M, Zhang M, Wright G, Huang DW, et al. Targeting the HTLV-I-regulated BATF3/IRF4 transcriptional network in adult T cell leukemia/lymphoma. Cancer Cell. 2018;34(2):286–97e10. https://doi.org/10.1016/j.ccell.2018.06.014. Chen D, Zhao Z, Huang Z, Chen DC, Zhu XX, Wang YZ, et al. Super enhancer inhibitors suppress MYC driven transcriptional amplification and tumor progression in osteosarcoma. Bone Res. 2018;6:11. https://doi.org/10.1038/s41413-018-0009-8. Li Y, Rivera CM, Ishii H, Jin F, Selvaraj S, Lee AY, et al. CRISPR reveals a distal super-enhancer required for Sox2 expression in mouse embryonic stem cells. PLoS One. 2014;9(12):e114485. https://doi.org/10.1371/journal.pone.0114485. Hnisz D, Schuijers J, Lin CY, Weintraub AS, Abraham BJ, Lee TI, et al. Convergence of developmental and oncogenic signaling pathways at transcriptional super-enhancers. Mol Cell. 2015;58(2):362–70. https://doi.org/10.1016/j.molcel.2015.02.014. Moorthy SD, Davidson S, Shchuka VM, Singh G, Malek-Gilani N, Langroudi L, et al. Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes. Genome Res. 2017;27(2):246–58. https://doi.org/10.1101/gr.210930.116. Dave K, Sur I, Yan J, Zhang J, Kaasinen E, Zhong F, et al. Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth. Elife. 2017;6. https://doi.org/10.7554/eLife.23382. Huang J, Li K, Cai W, Liu X, Zhang Y, Orkin SH, et al. Dissecting super-enhancer hierarchy based on chromatin interactions. Nat Commun. 2018;9(1):943. https://doi.org/10.1038/s41467-018-03279-9. Schuijers J, Manteiga JC, Weintraub AS, Day DS, Zamudio AV, Hnisz D, et al. Transcriptional dysregulation of MYC reveals common enhancer-docking mechanism. Cell Rep. 2018;23(2):349–60. https://doi.org/10.1016/j.celrep.2018.03.056. Pantera H, Moran JJ, Hung HA, Pak E, Dutra A, Svaren J. Regulation of the neuropathy-associated Pmp22 gene by a distal super-enhancer. Hum Mol Genet. 2018;27(16):2830–9. https://doi.org/10.1093/hmg/ddy191. Metser G, Shin HY, Wang C, Yoo KH, Oh S, Villarino AV, et al. An autoregulatory enhancer controls mammary-specific STAT5 functions. Nucleic Acids Res. 2016;44(3):1052–63. https://doi.org/10.1093/nar/gkv999. Zeng X, Willi M, Shin HY, Hennighausen L, Wang C. Lineage-specific and non-specific cytokine-sensing genes respond differentially to the master regulator STAT5. Cell Rep. 2016;17(12):3333–46. https://doi.org/10.1016/j.celrep.2016.11.079. Willi M, Yoo KH, Wang C, Trajanoski Z, Hennighausen L. Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation. Nucleic Acids Res. 2016;44(21):10277–91. https://doi.org/10.1093/nar/gkw844. Shin HY, Wang C, Lee HK, Yoo KH, Zeng X, Kuhns T, et al. CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome. Nat Commun. 2017;8:15464. https://doi.org/10.1038/ncomms15464. Sung YH, Kim JM, Kim HT, Lee J, Jeon J, Jin Y, et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Res. 2014;24(1):125–31. https://doi.org/10.1101/gr.163394.113. McVey M, Lee SE. MMEJ repair of double-strand breaks (director's cut): deleted sequences and alternative endings. Trends Genet. 2008;24(11):529–38. https://doi.org/10.1016/j.tig.2008.08.007. Zhou J, Wang J, Shen B, Chen L, Su Y, Yang J, et al. Dual sgRNAs facilitate CRISPR/Cas9-mediated mouse genome targeting. FEBS J. 2014;281(7):1717–25. https://doi.org/10.1111/febs.12735. Fujii W, Kawasaki K, Sugiura K, Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Res. 2013;41(20):e187. https://doi.org/10.1093/nar/gkt772. Wang L, Shao Y, Guan Y, Li L, Wu L, Chen F, et al. Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci Rep. 2015;5:17517. https://doi.org/10.1038/srep17517. Hara S, Kato T, Goto Y, Kubota S, Tamano M, Terao M, et al. Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system. J Reprod Dev. 2016;62(5):531–6. https://doi.org/10.1262/jrd.2016-058. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol. 2018;36(8):765–71. https://doi.org/10.1038/nbt.4192. Lee H, Kim JS. Unexpected CRISPR on-target effects. Nat Biotechnol. 2018;36(8):703–4. https://doi.org/10.1038/nbt.4207. Bolukbasi MF, Gupta A, Wolfe SA. Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat Methods. 2016;13(1):41–50. https://doi.org/10.1038/nmeth.3684. Kanchiswamy CN, Maffei M, Malnoy M, Velasco R, Kim JS. Fine-tuning next-generation genome editing tools. Trends Biotechnol. 2016;34(7):562–74. https://doi.org/10.1016/j.tibtech.2016.03.007. Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12(3):237–43, 1 p following 43. https://doi.org/10.1038/nmeth.3284. Crosetto N, Mitra A, Silva MJ, Bienko M, Dojer N, Wang Q, et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013;10(4):361–5. https://doi.org/10.1038/nmeth.2408. Wang X, Wang Y, Wu X, Wang J, Wang Y, Qiu Z, et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol. 2015;33(2):175–8. https://doi.org/10.1038/nbt.3127. Gabriel R, Lombardo A, Arens A, Miller JC, Genovese P, Kaeppel C, et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat Biotechnol. 2011;29(9):816–23. https://doi.org/10.1038/nbt.1948. Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33(2):179–86. https://doi.org/10.1038/nbt.3101. Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33(2):187–97. https://doi.org/10.1038/nbt.3117. Kim HK, Song M, Lee J, Menon AV, Jung S, Kang YM, et al. In vivo high-throughput profiling of CRISPR-Cpf1 activity. Nat Methods. 2017;14(2):153–9. https://doi.org/10.1038/nmeth.4104. Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, et al. In vivo genome editing with a small Cas9 orthologue derived from campylobacter jejuni. Nat Commun. 2017;8:14500. https://doi.org/10.1038/ncomms14500. Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM, et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods. 2015;12(12):1143–9. https://doi.org/10.1038/nmeth.3630. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013;154(2):442–51. https://doi.org/10.1016/j.cell.2013.06.044. Konermann S, Brigham MD, Trevino A, Hsu PD, Heidenreich M, Cong L, et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013;500(7463):472–6. https://doi.org/10.1038/nature12466. Hilton IB, D'Ippolito AM, Vockley CM, Thakore PI, Crawford GE, Reddy TE, et al. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol. 2015;33(5):510–7. https://doi.org/10.1038/nbt.3199. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013;10(10):973–6. https://doi.org/10.1038/nmeth.2600. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10(10):977–9. https://doi.org/10.1038/nmeth.2598. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013;31(9):833–8. https://doi.org/10.1038/nbt.2675. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature. 2016;533(7603):420–4. https://doi.org/10.1038/nature17946. Rees HA, Komor AC, Yeh WH, Caetano-Lopes J, Warman M, Edge ASB, et al. Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery. Nat Commun. 2017;8:15790. https://doi.org/10.1038/ncomms15790. Liang P, Sun H, Sun Y, Zhang X, Xie X, Zhang J, et al. Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell. 2017;8(8):601–11. https://doi.org/10.1007/s13238-017-0418-2. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, et al. Publisher correction: programmable base editing of a*T to G*C in genomic DNA without DNA cleavage. Nature. 2018;559:E8. https://doi.org/10.1038/s41586-018-0070-x. Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, et al. Base editing with a Cpf1-cytidine deaminase fusion. Nat Biotechnol. 2018;36(4):324–7. https://doi.org/10.1038/nbt.4102. Qu Y, Han B, Gao B, Bose S, Gong Y, Wawrowsky K, et al. Differentiation of human induced pluripotent stem cells to mammary-like organoids. Stem Cell Reports. 2017;8(2):205–15. https://doi.org/10.1016/j.stemcr.2016.12.023. Jamieson PR, Dekkers JF, Rios AC, Fu NY, Lindeman GJ, Visvader JE. Derivation of a robust mouse mammary organoid system for studying tissue dynamics. Development. 2017;144(6):1065–71. https://doi.org/10.1242/dev.145045. Laperrousaz B, Porte S, Gerbaud S, Harma V, Kermarrec F, Hourtane V, et al. Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens. Nucleic Acids Res. 2018;46(12):e70. https://doi.org/10.1093/nar/gky030. Hennighausen L. Mouse models for breast cancer. Breast Cancer Res. 2000;2(1):2–7. Pittius CW, Hennighausen L, Lee E, Westphal H, Nicols E, Vitale J, et al. A milk protein gene promoter directs the expression of human tissue plasminogen activator cDNA to the mammary gland in transgenic mice. Proc Natl Acad Sci U S A. 1988;85(16):5874–8. Liu X, Robinson GW, Wagner KU, Garrett L, Wynshaw-Boris A, Hennighausen L. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 1997;11(2):179–86. Wagner KU, Wall RJ, St-Onge L, Gruss P, Wynshaw-Boris A, Garrett L, et al. Cre-mediated gene deletion in the mammary gland. Nucleic Acids Res. 1997;25(21):4323–30. Boroviak K, Doe B, Banerjee R, Yang F, Bradley A. Chromosome engineering in zygotes with CRISPR/Cas9. Genesis. 2016;54(2):78–85. https://doi.org/10.1002/dvg.22915. Haapaniemi E, Botla S, Persson J, Schmierer B, Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med. 2018;24:927–30. https://doi.org/10.1038/s41591-018-0049-z. Ihry RJ, Worringer KA, Salick MR, Frias E, Ho D, Theriault K, et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med. 2018. https://doi.org/10.1038/s41591-018-0050-6.