An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

doi:10.1007/s13238-016-0360-8

Protein Cell

2017, Vol. 8

Issue (5) : 379-393 https://doi.org/10.1007/s13238-016-0360-8

RESEARCH ARTICLE

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Jianying Guo¹, Dacheng Ma², Rujin Huang¹, Jia Ming¹, Min Ye¹, Kehkooi Kee¹, Zhen Xie², Jie Na¹(

)

¹. Department of Basic Medical Sciences, School of Medicine, Center for Stem Cell Biology, Tsinghua University, Beijing 100084, China
². MOE Key Laboratory of Bioinformatics and Bioinformatics Division, Center for Synthetic and System Biology, TNLIST/Department of Automation, Tsinghua University, Beijing 100084, China

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Abstract

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Keywords CRISPR transcription activation human pluripotent stem cells NANOG pluripotency

Corresponding Author(s): Jie Na

Issue Date: 12 June 2017

Cite this article:

Jianying Guo,Dacheng Ma,Rujin Huang, et al. An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells[J]. Protein Cell, 2017, 8(5): 379-393.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-016-0360-8
https://academic.hep.com.cn/pac/EN/Y2017/V8/I5/379

1	BakerDEC, HarrisonNJ, MaltbyE, SmithK, MooreHD, ShawPJ, HeathPR, HoldenH, AndrewsPW (2007) Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol25:207–215 https://doi.org/10.1038/nbt1285
2	BalboaD, WeltnerJ, EurolaS, TrokovicR, WartiovaaraK, OtonkoskiT (2015) Conditionally stabilized dCas9 activator for controlling gene expression in human cell reprogramming and differentiation. Stem Cell Rep5:448–459 https://doi.org/10.1016/j.stemcr.2015.08.001
3	BedzhovI, Zernicka-GoetzM (2014) Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell156:1032–1044 https://doi.org/10.1016/j.cell.2014.01.023
4	BoyerLA, LeeTI, ColeMF, JohnstoneSE, LevineSS, ZuckerJP, GuentherMG, KumarRM, MurrayHL, JennerRG (2005) Core transcriptional regulatory circuitry in human embryonic stem cells. Cell122:947–956 https://doi.org/10.1016/j.cell.2005.08.020
5	BronsIGM, SmithersLE, TrotterMWB, Rugg-GunnP, SunB, de Sousa LopesSMC, HowlettSK, ClarksonA, Ahrlund-RichterL, PedersenRA (2007) Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature448:191–195 https://doi.org/10.1038/nature05950
6	BurridgePW, HolmströmA, and WuJC (2015). Chemically defined culture and cardiomyocyte differentiation of human pluripotent stem cells. Curr Protoc Hum Genet: 21.23. 21–21.23. 15 https://doi.org/10.1002/0471142905.hg2103s87
7	ChambersI, SilvaJ, ColbyD, NicholsJ, NijmeijerB, RobertsonM, VranaJ, JonesK, GrotewoldL, SmithA (2007) Nanog safeguards pluripotency and mediates germline development. Nature450:1230–1234 https://doi.org/10.1038/nature06403
8	ChangM-Y, RheeY-H, YiS-H, LeeS-J, KimR-K, KimH, ParkC-H, LeeS-H (2014) Doxycycline enhances survival and self-renewal of human pluripotent stem cells. Stem Cell Rep3:353–364 https://doi.org/10.1016/j.stemcr.2014.06.013
9	ChavezA, ScheimanJ, VoraS, PruittBW, TuttleM, IyerEPR, LinS, KianiS, GuzmanCD, WiegandDJ (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods12:326–328 https://doi.org/10.1038/nmeth.3312
10	ChenY, NiuY, LiY, AiZ, KangY, ShiH, XiangZ, YangZ, TanT, SiW (2015) Generation of cynomolgus monkey chimeric fetuses using embryonic stem cells. Cell Stem Cell17:116–124 https://doi.org/10.1016/j.stem.2015.06.004
11	DeglincertiA, CroftGF, PietilaLN, Zernicka-GoetzM, SiggiaED, BrivanlouAH (2016) Self-organization of the in vitro attached human embryo. Nature533:251–254 https://doi.org/10.1038/nature17948
12	DeKelverRC, ChoiVM, MoehleEA, PaschonDE, HockemeyerD, MeijsingSH, SancakY, CuiX, SteineEJ, MillerJC (2010) Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res20:1133–1142 https://doi.org/10.1101/gr.106773.110
13	DuggalG, WarrierS, GhimireS, BroekaertD, Van der JeughtM, LiermanS, DerooT, PeelmanL, Van SoomA, CornelissenR (2015) Alternative routes to induce naive pluripotency in human embryonic stem cells. Stem Cells33:2686–2698 https://doi.org/10.1002/stem.2071
14	GafniO, WeinbergerL, MansourAA, ManorYS, ChomskyE, Ben-YosefD, KalmaY, ViukovS, MazaI, ZviranA (2013) Derivation of novel human ground state naive pluripotent stem cells. Nature504:282–286 https://doi.org/10.1038/nature12745
15	GengaRM, KearnsNA, MaehrR (2016) Controlling transcription in human pluripotent stem cells using CRISPR-effectors. Methods101:36–42 https://doi.org/10.1016/j.ymeth.2015.10.014
16	GilbertLA, HorlbeckMA, AdamsonB, VillaltaJE, ChenY, WhiteheadEH, GuimaraesC, PanningB, PloeghHL, BassikMC (2014) Genome-scale CRISPR-mediated control of gene repression and activation. Cell159:647–661 https://doi.org/10.1016/j.cell.2014.09.029
17	GongS, LiQ, JeterCR, FanQ, TangDG, LiuB (2015) Regulation of NANOG in cancer cells. Mol Carcinog54:679–687 https://doi.org/10.1002/mc.22340
18	GonzálezF, ZhuZ, ShiZ-D, LelliK, VermaN, LiQV, HuangfuD (2014) An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell15:215–226 https://doi.org/10.1016/j.stem.2014.05.018
19	HannaJ, SahaK, PandoB, Van ZonJ, LengnerCJ, CreyghtonMP, van OudenaardenA, JaenischR (2009) Direct cell reprogramming is a stochastic process amenable to acceleration. Nature462:595–601 https://doi.org/10.1038/nature08592
20	HannaJ, ChengAW, SahaK, KimJ, LengnerCJ, SoldnerF, CassadyJP, MuffatJ, CareyBW, JaenischR (2010) Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc Natl Acad Sci107:9222–9227 https://doi.org/10.1073/pnas.1004584107
21	HiltonIB, D’IppolitoAM, VockleyCM, ThakorePI, CrawfordGE, ReddyTE, GersbachCA (2015) Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat Biotechnol33:510–517 https://doi.org/10.1038/nbt.3199
22	HockemeyerD, SoldnerF, BeardC, GaoQ, MitalipovaM, DeKelverRC, KatibahGE, AmoraR, BoydstonEA, ZeitlerB (2009) Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol27:851–857 https://doi.org/10.1038/nbt.1562
23	HoganB, CostantiniF, LacyE (1986) Manipulating the mouse embryo: a laboratory manual, vol 34. Cold spring harbor laboratory, Cold Spring Harbor, NY
24	HsuPD, LanderES, ZhangF (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell157:1262–1278 https://doi.org/10.1016/j.cell.2014.05.010
25	KearnsNA, GengaRMJ, EnuamehMS, GarberM, WolfeSA, MaehrR (2014) Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. Development141:219–223 https://doi.org/10.1242/dev.103341
26	KonermannS, BrighamMD, TrevinoAE, JoungJ, AbudayyehOO, BarcenaC, HsuPD, HabibN, GootenbergJS, NishimasuH (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature517:583–588 https://doi.org/10.1038/nature14136
27	LombardoA, CesanaD, GenoveseP, Di StefanoB, ProvasiE, ColomboDF, NeriM, MagnaniZ, CantoreA, RisoPL (2011) Sitespecific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods8:861–869 https://doi.org/10.1038/nmeth.1674
28	MaederML, LinderSJ, CascioVM, FuY, HoQH, JoungJK (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods10:977–979 https://doi.org/10.1038/nmeth.2598
29	MandegarMA, HuebschN, FrolovEB, ShinE, TruongA, OlveraMP, ChanAH, MiyaokaY, HolmesK, SpencerCI (2016) CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell18:541–553 https://doi.org/10.1016/j.stem.2016.01.022
30	MitsuiK, TokuzawaY, ItohH, SegawaK, MurakamiM, TakahashiK, MaruyamaM, MaedaM, YamanakaS (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell113:631–642 https://doi.org/10.1016/S0092-8674(03)00393-3
31	OrdovásL, BoonR, PistoniM, ChenY,,WolfsE, GuoW, SambathkumarR, Bobis-WozowiczS, HelsenN, VanhoveJ (2015) Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locusmediated transgene inhibition. Stem cell Rep5:918–931 https://doi.org/10.1016/j.stemcr.2015.09.004
32	QianK, HuangCL, ChenH, BlackbournLW, ChenY, CaoJ, YaoL, SauveyC, DuZ, ZhangSC (2014) A simple and efficient system for regulating gene expression in human pluripotent stem cells and derivatives. Stem Cells32:1230–1238 https://doi.org/10.1002/stem.1653
33	ShahbaziMN, JedrusikA, VuoristoS, RecherG, HupalowskaA, BoltonV, FogartyNME, CampbellA, DevitoLG, IlicD (2016) Selforganization of the human embryo in the absence of maternal tissues. Nature cell Biol18:700–708 https://doi.org/10.1038/ncb3347
34	SilvaJ, NicholsJ, TheunissenTW, GuoG, van OostenAL, BarrandonO, WrayJ, YamanakaS, ChambersI, SmithA (2009) Nanog is the gateway to the pluripotent ground state. Cell138:722–737 https://doi.org/10.1016/j.cell.2009.07.039
35	SmithJR, MaguireS, DavisLA, AlexanderM, YangF, ChandranS, PedersenRA(2008) Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells26:496–504 https://doi.org/10.1634/stemcells.2007-0039
36	TaapkenSM, NislerBS, NewtonMA, Sampsell-BarronTL, LeonhardKA, McIntireEM, MontgomeryKD (2011) Karyotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat Biotechnol29:313–314 https://doi.org/10.1038/nbt.1835
37	TakashimaY, GuoG, LoosR, NicholsJ, FiczG, KruegerF, OxleyD, SantosF, ClarkeJ, MansfieldW (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell158:1254–1269 https://doi.org/10.1016/j.cell.2014.08.029
38	TanenbaumME, GilbertLA, QiLS, WeissmanJS, ValeRD (2014) A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell159:635–646 https://doi.org/10.1016/j.cell.2014.09.039
39	TesarPJ, ChenowethJG, BrookFA, DaviesTJ, EvansEP, MackDL, GardnerRL, McKayRDG (2007) New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature448:196–199 https://doi.org/10.1038/nature05972
40	TheunissenTW, PowellBE, WangH, MitalipovaM, FaddahDA, ReddyJ, FanZP, MaetzelD, GanzK, ShiL (2014) Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell15:471–487 https://doi.org/10.1016/j.stem.2014.07.002
41	WareCB, NelsonAM, MechamB, HessonJ, ZhouW, JonlinEC, Jimenez-CalianiAJ, DengX, CavanaughC, CookS (2014) Derivation of naive human embryonic stem cells. Proc Natl Acad Sci111:4484–4489 https://doi.org/10.1073/pnas.1319738111
42	WiedenheftB, SternbergSH, DoudnaJA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature482:331–338 https://doi.org/10.1038/nature10886
43	XuX, TaoY, GaoX, ZhangL, LiX, ZouW, RuanK, WangF, G-lXu, HuR (2016) A CRISPR-based approach for targeted DNA demethylation. Cell Discov2:16009 https://doi.org/10.1038/celldisc.2016.9
44	ZhuZ, GonzálezF, HuangfuD (2014) The iCRISPR platform for rapid genome editing in human pluripotent stem cells. Methods Enzymol546:215 https://doi.org/10.1016/B978-0-12-801185-0.00011-8
45	ZhuZ, VermaN, GonzálezF, ShiZ-D, HuangfuD (2015) A CRISPR/Cas-mediated selection-free knockin strategy in human embryonic stem cells. Stem Cell Rep4:1103–1111 https://doi.org/10.1016/j.stemcr.2015.04.016

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