The last decade has seen many exciting technological breakthroughs that greatly expanded the toolboxes for biological and biomedical research, yet few have had more impact than induced pluripotent stem cells and modern-day genome editing. These technologies are providing unprecedented opportunities to improve physiological relevance of experimental models, further our understanding of developmental processes, and develop novel therapies. One of the research areas that benefit greatly from these technological advances is the three-dimensional human organoid culture systems that resemble human tissues morphologically and physiologically. Here we summarize the development of human pluripotent stem cells and their differentiation through organoid formation. We further discuss how genetic modifications, genome editing in particular, were applied to answer basic biological and biomedical questions using organoid cultures of both somatic and pluripotent stem cell origins. Finally, we discuss the potential challenges of applying human pluripotent stem cell and organoid technologies for safety and efficiency evaluation of emerging genome editing tools.
WuJIzpisua BelmonteJC.Stem cells: a renaissance in human biology research.Cell2016;
165:1572–85
2.
ThomsonJItskovitz-EldorJShapiroSWaknitzMSwiergielJMarshallVJonesJ.Embryonic stem cell lines derived from human blastocysts. Science1998;
282:1145–7
3.
DrukkerMBenvenistyN.The immunogenicity of human embryonic stem-derived cells. Trends Biotechnol2004;
22:136–41
4.
BradleyJBoltonEPedersenR.Stem cell medicine encounters the immune system. Nat Rev Immunol2002;
2:859–71
5.
TakahashiKYamanakaS.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell2006;
126:663–76
6.
TakahashiKTanabeKOhnukiMNaritaMIchisakaTTomodaKYamanakaS.Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell2007;
131:861–72
7.
JinekMChylinskiKFonfaraIHauerMDoudnaJACharpentierE.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science2012;
337:816–21
8.
ThomasKRCapecchiMR.Introduction of homologous DNA sequences into mammalian cells induces mutations in the cognate gene. Nature1986;
324:34–8
9.
EvansMKaufmanM.Establishment in culture of pluripotential cells from mouse embryos. Nature1981;
292:154–6
10.
DoetschmanTGreggRGMaedaNHooperMLMeltonDWThompsonSSmithiesO.Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature1987;
330:576–8
11.
RevazovaETurovetsNKochetkovaOKindarovaLKuzmichevLJanusJPryzhkovaM.Patient-specific stem cell lines derived from human parthenogenetic blastocysts. Cloning Stem Cells2007;
9:432–49
12.
MaiQYuYLiTWangLChenMJHuangSZZhouCZhouQ.Derivation of human embryonic stem cell lines from parthenogenetic blastocysts. Cell Res2007;
17:1008–19
13.
LinGOuYangQZhouXGuYYuanDLiWLiuGLiuTLuG.A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure. Cell Res2007;
17:999–1007
de WertGMummeryC.Human embryonic stem cells: research, ethics and policy. Hum Reprod2003;
18:672–82
16.
ParkIZhaoRWestJYabuuchiAHuoHInceTLerouPLenschMDaleyG.Reprogramming of human somatic cells to pluripotency with defined factors. Nature2008;
451:141–6
17.
KimJZaehresHWuGGentileLKoKSebastianoVAraúzo-BravoMRuauDHanDZenkeMSchölerH.Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature2008;
454:646–50
18.
AasenTRayaABarreroMGarretaEConsiglioAGonzalezFVassenaRBilićJPekarikVTiscorniaGEdelMBouéSBelmonteJ.Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol2008;
26:1276–84
19.
HaaseAOlmerRSchwankeKWunderlichSMerkertSHessCZweigerdtRGruhIMeyerJWagnerSMaierLHanDGlageSMillerKFischerPSchölerHMartinU.Generation of induced pluripotent stem cells from human cord blood. Cell Stem Cell2009;
5:434–41
20.
GiorgettiAMontserratNAasenTGonzalezFRodríguez-PizàIVassenaRRayaABouéSBarreroMCorbellaBTorrabadellaMVeigaAIzpisua BelmonteJ.Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell2009;
5:353–7
21.
LohYAgarwalSParkIUrbachAHuoHHeffnerGKimKMillerJNgKDaleyG.Generation of induced pluripotent stem cells from human blood. Blood2009;
113:5476–9
22.
YeZZhanHMaliPDoweySWilliamsDMJangYYDangCVSpivakJLMoliternoARChengL.Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders. Blood2009;
114:5473–80
23.
BrownMERondonERajeshDMackALewisRFengXZiturLJLearishRDNuwaysirEF.Derivation of induced pluripotent stem cells from human peripheral blood T lymphocytes. PLoS One2010;
5:e11373
24.
StaerkJDawlatyMMGaoQMaetzelDHannaJSommerCAMostoslavskyG.JaenischR.Reprogramming of human peripheral blood cells to induced pluripotent stem cells. Cell Stem Cell2010;
7:20–4
25.
ChoiSMLiuHChaudhariPKimYChengLFengJSharkisSYeZJangYY.Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells. Blood2011;
118:1801–5
26.
LiuHYeZKimYSharkisSJangYY.Generation of endoderm-derived human induced pluripotent stem cells from primary hepatocytes. Hepatology2010;
51:1810–9
27.
YuJVodyanikMSmuga-OttoKAntosiewicz-BourgetJFraneJTianSNieJJonsdottirGRuottiVStewartRSlukvinIThomsonJ.Induced pluripotent stem cell lines derived from human somatic cells. Science2007;
318:1917–20
KotiniAGChangCJBoussaadIDelrowJJDolezalEKNagulapallyABPernaFFishbeinGAKlimekVMHawkinsRDHuangfuDMurryCEGraubertTNimerSDPapapetrouEP.Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells. Nat Biotechnol2015;
33:646–55
30.
ChaoMPGentlesAJChatterjeeSLanFReinischACorcesMRXavySShenJHaagDChandaSSinhaRMorgantiRMNishimuraTAmeenMWuHWernigMWuJCMajetiR.Human AML-iPSCs reacquire leukemic properties after differentiation and model clonal variation of disease. Cell Stem Cell2017;
20:329–44.e7
31.
LiangGZhangY.Genetic and epigenetic variations in iPSCs: potential causes and implications for application. Cell Stem Cell2013;
13:149–59
32.
NishizawaMChonabayashiKNomuraMTanakaANakamuraMInagakiANishikawaMTakeiIOishiATanabeKOhnukiMYokotaHKoyanagi-AoiMOkitaKWatanabeATakaori-KondoAYamanakaSYoshidaY.Epigenetic variation between human induced pluripotent stem cell lines is an indicator of differentiation capacity. Cell Stem Cell2016;
19:341–54
EirakuMWatanabeKMatsuo-TakasakiMKawadaMYonemuraSMatsumuraMWatayaTNishiyamaAMugurumaKSasaiY.Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell2008;
3:519–32
35.
EstesJDWongSWBrenchleyJM.Nonhuman primate models of human viral infections. Nat Rev Immunol2018;
18:390–404
36.
AnisimovVNUkraintsevaSVYashinAI.Cancer in rodents: does it tell us about cancer in humans?Nat Rev Cancer2005;
5:807–19
37.
JakelRJSchneiderBLSvendsenCN.Using human neural stem cells to model neurological disease. Nat Rev Genet2004;
5:136–44
38.
IefremovaVManikakisGKrefftOJabaliAWeynansKWilkensRMarsonerFBrandlBMullerFJKochPLadewigJ.An organoid-based model of cortical development identifies non-cell-autonomous defects in wnt signaling contributing to Miller-Dieker syndrome. Cell Rep2017;
19:50–9
39.
BershteynMNowakowskiTJPollenAADi LulloENeneAWynshaw-BorisAKriegsteinAR.Human iPSC-derived cerebral organoids model cellular features of lissencephaly and reveal prolonged mitosis of outer radial glia. Cell Stem Cell2017;
20:435–49 e4
40.
LinkousABalamatsiasDSnuderlMEdwardsLMiyaguchiKMilnerTReichBCohen-GouldLStoraskaANakayamaYSchenkeinESinghaniaRCiriglianoSMagdeldinTLinYNanjangudGChadalavadaKPisapiaDListonCFineHA.Modeling patient-derived glioblastoma with cerebral organoids. Cell Rep2019;
26:3203–11.e5
41.
XiangYTanakaYCakirBPattersonBKimKYSunPKangYJZhongMLiuXPatraPLeeSHWeissmanSMParkIH.hESC-derived thalamic organoids form reciprocal projections when fused with cortical organoids. Cell Stem Cell2019;
24:487–97 e7
42.
ParfittDALaneARamsdenCMCarrAJMunroPMJovanovicKSchwarzNKanugaNMuthiahMNHullSGalloJMda CruzLMooreATHardcastleAJCoffeyPJCheethamME.Identification and correction of mechanisms underlying inherited blindness in human iPSC-derived optic cups. Cell Stem Cell2016;
18:769–81
43.
LiYMuffatJOmerABoschILancasterMASurMGehrkeLKnoblichJAJaenischR.Induction of expansion and folding in human cerebral organoids. Cell Stem Cell2017;
20:385–96.e3
44.
NakanoTAndoSTakataNKawadaMMugurumaKSekiguchiKSaitoKYonemuraSEirakuMSasaiY.Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell2012;
10:771–85
45.
TakebeTSekineKKimuraMYoshizawaEAyanoSKoidoMFunayamaSNakanishiNHisaiTKobayashiTKasaiTKitadaRMoriAAyabeHEjiriYAmimotoNYamazakiYOgawaSIshikawaMKiyotaYSatoYNozawaKOkamotoSUenoYTaniguchiH.Massive and reproducible production of liver buds entirely from human pluripotent stem cells. Cell Rep2017;
21:2661–70
46.
MiuraSSuzukiA.Generation of mouse and human organoid-forming intestinal progenitor cells by direct lineage reprogramming. Cell Stem Cell2017;
21:456–71 e5
47.
MithalACapillaAHeinzeDBericalAVillacorta-MartinCVedaieMJacobAAboKSzymaniakAPeasleyMStufferAMahoneyJKottonDNHawkinsFMostoslavskyG.Generation of mesenchyme free intestinal organoids from human induced pluripotent stem cells. Nat Commun2020;
11:215
48.
MuneraJOSundaramNRankinSAHillDWatsonCMaheMVallanceJEShroyerNFSinagogaKLZarzoso-LacosteAHudsonJRHowellJCChatuvediPSpenceJRShannonJMZornAMHelmrathMAWellsJM.Differentiation of human pluripotent stem cells into colonic organoids via transient activation of BMP signaling. Cell Stem Cell2017;
21:51–64.e6
49.
DyeBRYoungbloodRLOakesRSKasputisTCloughDWSpenceJRSheaLD.Human lung organoids develop into adult airway-like structures directed by physico-chemical biomaterial properties. Biomaterials2020;
234:119757
50.
ChenYWHuangSXde CarvalhoAHoSHIslamMNVolpiSNotarangeloLDCiancanelliMCasanovaJLBhattacharyaJLiangAFPalermoLMPorottoMMosconaASnoeckHW.A three-dimensional model of human lung development and disease from pluripotent stem cells. Nat Cell Biol2017;
19:542–9
51.
MillerAJHillDRNagyMSAokiYDyeBRChinAMHuangSZhuFWhiteESLamaVSpenceJR.In vitro induction and in vivo engraftment of lung bud tip progenitor cells derived from human pluripotent stem cells. Stem Cell Rep2018;
10:101–19
52.
McCauleyKBHawkinsFSerraMThomasDCJacobAKottonDN.Efficient derivation of functional human airway epithelium from pluripotent stem cells via temporal regulation of wnt signaling. Cell Stem Cell2017;
20:844–57 e6
MillsRJParkerBLQuaife-RyanGAVogesHKNeedhamEJBornotADingMAnderssonHPollaMElliottDADrowleyLClausenMPlowrightATBarrettIPWangQDJamesDEPorrelloERHudsonJE.Drug screening in human PSC-cardiac organoids identifies pro-proliferative compounds acting via the mevalonate pathway. Cell Stem Cell2019;
24:895–907.e6
55.
TianLDeshmukhAYeZJangYY.Efficient and controlled generation of 2D and 3D bile duct tissue from human pluripotent stem cell-derived spheroids. Stem Cell Rev Rep2016;
12:500–8
56.
MunSJRyuJSLeeMOSonYSOhSJChoHSSonMYKimDSKimSJYooHJLeeHJKimJJungCRChungKSSonMJ.Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids. J Hepatol2019;
71:970–85
57.
TakeishiKCollin de l'HortetAWangYHandaKGuzman-LepeJMatsubaraKMoritaKJangSHaepNFlorentinoRMYuanFFukumitsuKTobitaKSunWFranksJDelgadoERShapiroEMFraunhofferNADuncanAWYagiHMashimoTFoxIJSoto-GutierrezA.Assembly and function of a bioengineered human liver for transplantation generated solely from induced pluripotent stem cells. Cell Rep2020;
31:107711
58.
SunLWangYCenJMaXCuiLQiuZZhangZLiHYangRZWangCChenXWangLYeYZhangHPanGKangJSJiYZhengYWZhengSHuiL.Modelling liver cancer initiation with organoids derived from directly reprogrammed human hepatocytes. Nat Cell Biol2019;
21:1015–26
59.
TakebeTSekineKEnomuraMKoikeHKimuraMOgaeriTZhangRRUenoYZhengYWKoikeNAoyamaSAdachiYTaniguchiH.Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature2013;
499:481–4
60.
YoshiharaEO'ConnorCGasserEWeiZOhTGTsengTWWangDCayabyabFDaiYYuRTLiddleCAtkinsARDownesMEvansRM.Immune-evasive human islet-like organoids ameliorate diabetes. Nature2020;
586:606–11
61.
HohwielerMIllingAHermannPCMayerTStockmannMPerkhoferLEiselerTAntonyJSMullerMRenzSKuoCCLinQSendlerMBreunigMKleidermanSMLechelAZenkerMLeichsenringMRosendahlJZenkeMSainzBJr.MayerleJCostaIGSeufferleinTKormannMWagnerMLiebauSKlegerA.Human pluripotent stem cell-derived acinar/ductal organoids generate human pancreas upon orthotopic transplantation and allow disease modelling. Gut2017;
66:473–86
62.
PhipsonBErPXCombesANForbesTAHowdenSEZappiaLYenHJLawlorKTHaleLJSunJWolvetangETakasatoMOshlackALittleMH.Evaluation of variability in human kidney organoids. Nat Methods2019;
16:79–87
63.
TakasatoMErPXChiuHSMaierBBaillieGJFergusonCPartonRGWolvetangEJRoostMSChuva de Sousa LopesSMLittleMH.Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis. Nature2015;
526:564–8
64.
LowJHLiPChewEGYZhouBSuzukiKZhangTLianMMLiuMAizawaERodriguez EstebanCYongKSMChenQCampistolJMFangMKhorCCFooJNIzpisua BelmonteJCXiaY.Generation of human PSC-Derived kidney organoids with patterned nephron segments and a de novo vascular network. Cell Stem Cell2019;
25:373–87 e9
65.
ArtegianiBCleversH.Use and application of 3D-organoid technology. Hum Mol Genet2018;
27:R99–R107
66.
DrostJCleversH.Organoids in cancer research. Nat Rev Cancer2018;
18:407–18
67.
RossiGManfrinALutolfMP.Progress and potential in organoid research. Nat Rev Genet2018;
19:671–87
68.
LittleMHCombesAN.Kidney organoids: accurate models or fortunate accidents. Genes Dev2019;
33:1319–45
69.
BhaduriAAndrewsMGKriegsteinARNowakowskiTJ.Are organoids ready for prime time?Cell Stem Cell2020;
27:361–5
70.
Del DossoAUrendaJPNguyenTQuadratoG.Upgrading the physiological relevance of human brain organoids. Neuron2020;
107:1014–28
71.
KimJKooBKKnoblichJA.Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol2020;
21:571–84
72.
TuvesonDCleversH.Cancer modeling meets human organoid technology. Science2019;
364:952–5
73.
MonteilVKwonHPradoPHagelkruysAWimmerRAStahlMLeopoldiAGarretaEHurtado Del PozoCProsperFRomeroJPWirnsbergerGZhangHSlutskyASConderRMontserratNMirazimiAPenningerJM.Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell2020;
181:905–13 e7
74.
LamersMMBeumerJvan der VaartJKnoopsKPuschhofJBreugemTIRavelliRBGPaul van SchayckJMykytynAZDuimelHQvan DonselaarERieseboschSKuijpersHJHSchipperDvan de WeteringWJde GraafMKoopmansMCuppenEPetersPJHaagmansBLCleversH.SARS-CoV-2 productively infects human gut enterocytes. Science2020;
369:50–4
75.
ZhouJLiCLiuXChiuMCZhaoXWangDWeiYLeeAZhangAJChuHCaiJPYipCCChanIHWongKKTsangOTChanKHChanJFToKKChenHYuenKY.Infection of bat and human intestinal organoids by SARS-CoV-2. Nat Med2020;
26:1077–83
76.
YangLHanYNilsson-PayantBEGuptaVWangPDuanXTangXZhuJZhaoZJaffreFZhangTKimTWHarschnitzORedmondDHoughtonSLiuCNajiACiceriGGuttikondaSBramYNguyenDTCioffiMChandarVHoaglandDAHuangYXiangJWangHLydenDBorczukAChenHJStuderLPanFCHoDDtenOeverBREvansTSchwartzREChenS.A human pluripotent stem cell-based platform to study SARS-CoV-2 tropism and model virus infection in human cells and organoids. Cell Stem Cell2020;
27:125–36.e7
77.
JacobFPatherSRHuangWKZhangFWongSZHZhouHCubittBFanWChenCZXuMPradhanMZhangDYZhengWBangAGSongHCarlos de la TorreJMingGL.Human pluripotent stem Cell-Derived neural cells and brain organoids reveal SARS-CoV-2 neurotropism predominates in choroid plexus epithelium. Cell Stem Cell2020;
27:937–50
78.
RamaniAMullerLOstermannPNGabrielEAbida-IslamPMuller-SchiffmannAMariappanAGoureauOGruellHWalkerAAndreeMHaukaSHouwaartTDiltheyAWohlgemuthKOmranHKleinFWieczorekDAdamsOTimmJKorthCSchaalHGopalakrishnanJ.SARS-CoV-2 targets neurons of 3D human brain organoids. EMBO J2020;
39:e106230
79.
ZhangBZChuHHanSShuaiHDengJHuYFGongHRLeeACZouZYauTWuWHungIFChanJFYuenKYHuangJD.SARS-CoV-2 infects human neural progenitor cells and brain organoids. Cell Res2020;
30:928–31
80.
RamaniSCrawfordSEBluttSEEstesMK.Human organoid cultures: transformative new tools for human virus studies. Curr Opin Virol2018;
29:79–86
81.
ZwakaTThomsonJ.Homologous recombination in human embryonic stem cells. Nat Biotechnol2003;
21:319–21
82.
UrbachASchuldinerMBenvenistyN.Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells. Stem Cells2004;
22:635–41
83.
RouetPSmihFJasinM.Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol Cell Biol1994;
14:8096–106
84.
KimYGChaJChandrasegaranS.Hybrid restriction enzymes: zinc finger fusions to fok I cleavage domain. Proc Natl Acad Sci U S A1996;
93:1156–60
85.
SmithJBibikovaMWhitbyFGReddyARChandrasegaranSCarrollD.Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res2000;
28:3361–9
86.
BibikovaMCarrollDSegalDJTrautmanJKSmithJKimYGChandrasegaranS.Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol2001;
21:289–97
87.
ZouJMaederMMaliPPruett-MillerSThibodeau-BegannySChouBChenGYeZParkIDaleyGPorteusMJoungJChengL.Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell2009;
5:97–110
88.
HockemeyerDSoldnerFBeardCGaoQMitalipovaMDeKelverRKatibahGAmoraRBoydstonEZeitlerBMengXMillerJZhangLRebarEGregoryPUrnovFJaenischR.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol2009;
27:851–7
89.
BochJScholzeHSchornackSLandgrafAHahnSKaySLahayeTNickstadtABonasU.Breaking the code of DNA binding specificity of TAL-type III effectors. Science2009;
326:1509–12
90.
MoscouMJBogdanoveAJ.A simple cipher governs DNA recognition by TAL effectors. Science2009;
326:1501
91.
ChristianMCermakTDoyleELSchmidtCZhangFHummelABogdanoveAJVoytasDF.Targeting DNA double-strand breaks with TAL effector nucleases. Genetics2010;
186:757–61
92.
CermakTDoyleELChristianMWangLZhangYSchmidtCBallerJASomiaNVBogdanoveAJVoytasDF.Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res2011;
39:e82
93.
JoungJKSanderJD.TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol2013;
14:49–55
94.
HockemeyerDWangHKianiSLaiCSGaoQCassadyJPCostGJZhangLSantiagoYMillerJCZeitlerBCheroneJMMengXHinkleySJRebarEJGregoryPDUrnovFDJaenischR.Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol2011;
29:731–4
95.
ChoiSMKimYShimJSParkJTWangRHLeachSDLiuJODengCYeZJangYY.Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells. Hepatology2013;
57:2458–68
96.
MojicaFJFerrerCJuezGRodriguez-ValeraF.Long stretches of short tandem repeats are present in the largest replicons of the archaea Haloferax mediterranei and Haloferax volcanii and could be involved in replicon partitioning. Mol Microbiol1995;
17:85–93
97.
JansenREmbdenJDGaastraWSchoulsLM.Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol2002;
43:1565–75
98.
DeltchevaEChylinskiKSharmaCMGonzalesKChaoYPirzadaZAEckertMRVogelJCharpentierE.CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature2011;
471:602–7
99.
MaliPYangLEsveltKMAachJGuellMDiCarloJENorvilleJEChurchGM.RNA-guided human genome engineering via Cas9. Science2013;
339:823–6
100.
CongLRanFACoxDLinSBarrettoRHabibNHsuPDWuXJiangWMarraffiniLAZhangF.Multiplex genome engineering using CRISPR/cas systems. Science2013;
339:819–23
101.
ZetscheBGootenbergJSAbudayyehOOSlaymakerIMMakarovaKSEssletzbichlerPVolzSEJoungJvan der OostJRegevAKooninEVZhangF.Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell2015;
163:759–71
102.
AbudayyehOOGootenbergJSEssletzbichlerPHanSJoungJBelantoJJVerdineVCoxDBTKellnerMJRegevALanderESVoytasDFTingAYZhangF.RNA targeting with CRISPR-Cas13. Nature2017;
550:280–4
103.
LiuJJOrlovaNOakesBLMaESpinnerHBBaneyKLMChuckJTanDKnottGJHarringtonLBAl-ShayebBWagnerABrotzmannJStaahlBTTaylorKLDesmaraisJNogalesEDoudnaJA.CasX enzymes comprise a distinct family of RNA-guided genome editors. Nature2019;
566:218–23
104.
SmithCAbalde-AtristainLHeCBrodskyBRBraunsteinEMChaudhariPJangYYChengLYeZ.Efficient and allele-specific genome editing of disease loci in human iPSCs. Mol Ther2015;
23:570–7
105.
SmithCJCastanonOSaidKVolfVKhoshakhlaghPHornickAFerreiraRWuCTGuellMGargSNgAHMMyllykallioHChurchGM.Enabling large-scale genome editing at repetitive elements by reducing DNA nicking. Nucleic Acids Res2020;
48:5183–95
106.
KomorACKimYBPackerMSZurisJALiuDR.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature2016;
533:420–4
107.
GaudelliNMKomorACReesHAPackerMSBadranAHBrysonDILiuDR.Programmable base editing of a•T to G•C in genomic DNA without DNA cleavage. Nature2017;
551:464–71
108.
ReesHALiuDR.Base editing: precision chemistry on the genome and transcriptome of living cells. Nat Rev Genet2018;
19:770–88
109.
AnzaloneAVRandolphPBDavisJRSousaAAKoblanLWLevyJMChenPJWilsonCNewbyGARaguramALiuDR.Search-and-replace genome editing without double-strand breaks or donor DNA. Nature2019;
576:149–57
110.
DriehuisECleversH.CRISPR/cas 9 genome editing and its applications in organoids. Am J Physiol Gastrointest Liver Physiol2017;
312:G257–G65
111.
ArtegianiBHendriksDBeumerJKokRZhengXJooreIChuva de Sousa LopesSvan ZonJTansSCleversH.Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. Nat Cell Biol2020;
22:321–31
112.
WooDHChenQYangTLGlineburgMRHogeCLeuNAJohnsonFBLengnerCJ.Enhancing a Wnt-Telomere feedback loop restores intestinal stem cell function in a human organotypic model of dyskeratosis congenita. Cell Stem Cell2016;
19:397–405
113.
KhanTARevahOGordonAYoonSJKrawiszAKGooldCSunYKimCHTianYLiMYSchaepeJMIkedaKAminNDSakaiNYazawaMKushanLNishinoSPorteusMHRapoportJLBernsteinJAO'HaraRBeardenCEHallmayerJFHuguenardJRGeschwindDHDolmetschREPascaSP.Neuronal defects in a human cellular model of 22q11.2 deletion syndrome. Nat Med2020;
26:1888–98
114.
DengWLGaoMLLeiXLLvJNZhaoHHeKWXiaXXLiLYChenYCLiYPPanDXueTJinZB.Gene correction reverses ciliopathy and photoreceptor loss in iPSC-Derived retinal organoids from retinitis pigmentosa patients. Stem Cell Reports2018;
10:1267–81
115.
FreedmanBSBrooksCRLamAQFuHMorizaneRAgrawalVSaadAFLiMKHughesMRWerffRVPetersDTLuJBacceiASiedleckiAMValeriusMTMusunuruKMcNagnyKMSteinmanTIZhouJLerouPHBonventreJV.Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat Commun2015;
6:8715
116.
SchwankGKooBKSasselliVDekkersJFHeoIDemircanTSasakiNBoymansSCuppenEvan der EntCKNieuwenhuisEEBeekmanJMCleversH.Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell Stem Cell2013;
13:653–8
117.
BlairJDHockemeyerDBateupHS.Genetically engineered human cortical spheroid models of tuberous sclerosis. Nat Med2018;
24:1568–78
118.
ForbesTAHowdenSELawlorKPhipsonBMaksimovicJHaleLWilsonSQuinlanCHoGHolmanKBennettsBCrawfordJTrnkaPOshlackAPatelCMallettASimonsCLittleMH.Patient-iPSC-derived kidney organoids show functional validation of a ciliopathic renal phenotype and reveal underlying pathogenetic mechanisms. Am J Hum Genet2018;
102:816–31
119.
FujiiMMatanoMNankiKSatoT.Efficient genetic engineering of human intestinal organoids using electroporation. Nat Protoc2015;
10:1474–85
120.
MatanoMDateSShimokawaMTakanoAFujiiMOhtaYWatanabeTKanaiTSatoT.Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med2015;
21:256–62
121.
DrostJvan JaarsveldRHPonsioenBZimberlinCvan BoxtelRBuijsASachsNOvermeerRMOfferhausGJBegthelHKorvingJvan de WeteringMSchwankGLogtenbergMCuppenESnippertHJMedemaJPKopsGJCleversH.Sequential cancer mutations in cultured human intestinal stem cells. Nature2015;
521:43–7
122.
VerissimoCSOvermeerRMPonsioenBDrostJMertensSVerlaan-KlinkIGerwenBVvan der VenMWeteringMVEganDABernardsRCleversHBosJLSnippertHJ.Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening. Elife2016;
5:e18489
123.
DrostJvan BoxtelRBlokzijlFMizutaniTSasakiNSasselliVde LigtJBehjatiSGrollemanJEvan WezelTNik-ZainalSKuiperRPCuppenECleversH.Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. Science2017;
358:234–8
124.
YanHHNSiuHCHoSLYueSSKGaoYTsuiWYChanDChanASWongJWHManAHYLeeBCHChanASYChanAKWHuiHSCheungAKLLawWLLoOSHYuenSTCleversHLeungSY.Organoid cultures of early-onset colorectal cancers reveal distinct and rare genetic profiles. Gut2020;
69:2165–79
125.
OgawaJPaoGMShokhirevMNVermaIM.Glioblastoma model using human cerebral organoids. Cell Rep2018;
23:1220–9
SeinoTKawasakiSShimokawaMTamagawaHToshimitsuKFujiiMOhtaYMatanoMNankiKKawasakiKTakahashiSSugimotoSIwasakiETakagiJItoiTKitagoMKitagawaYKanaiTSatoT.Human pancreatic tumor organoids reveal loss of stem cell niche factor dependence during disease progression. Cell Stem Cell2018;
22:454–67.e6
128.
LiuXChengYAbrahamJMWangZWangZKeXYanRShinEJNgamruengphongSKhashabMAZhangGMcNamaraGEwaldAJLinDLiuZMeltzerSJ.Modeling Wnt signaling by CRISPR-Cas9 genome editing recapitulates neoplasia in human Barrett epithelial organoids. Cancer Lett2018;
436:109–18
129.
KawasakiKFujiiMSugimotoSIshikawaKMatanoMOhtaYToshimitsuKTakahashiSHosoeNSekineSKanaiTSatoT.Chromosome engineering of human colon-derived organoids to develop a model of traditional serrated adenoma. Gastroenterology2020;
158:638–51 e8
130.
IhryRJWorringerKASalickMRFriasEHoDTheriaultKKommineniSChenJSondeyMYeCRandhawaRKulkarniTYangZMcAllisterGRussCReece-HoyesJForresterWHoffmanGRDolmetschRKaykasA.p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med2018;
24:939–46
131.
HaapaniemiEBotlaSPerssonJSchmiererBTaipaleJ.CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med2018;
24:927–30
132.
GeurtsMHde PoelEAmatngalimGDOkaRMeijersFMKruisselbrinkEvan MourikPBerkersGde Winter-de GrootKMMichelSMuilwijkDAalbersBLMullendersJBojSFSuenSWFBrunsveldJEJanssensHMMallMAGraeberSYvan BoxtelRvan der EntCKBeekmanJMCleversH.CRISPR-based adenine editors correct nonsense mutations in a cystic fibrosis organoid biobank. Cell Stem Cell2020;
26:503–10.e7
133.
HuJHMillerSMGeurtsMHTangWChenLSunNZeinaCMGaoXReesHALinZLiuDR.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature2018;
556:57–63
134.
KleinstiverBPSousaAAWaltonRTTakYEHsuJYClementKWelchMMHorngJEMalagon-LopezJScarfòIMausMVPinelloLAryeeMJJoungJK.Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol2019;
37:276–82
135.
WaltonRTChristieKAWhittakerMNKleinstiverBP.Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science2020;
368:290–6
136.
ChatterjeePJakimoNLeeJAmraniNRodriguezTKosekiSRTTysingerEQingRHaoSSontheimerEJJacobsonJ.An engineered ScCas9 with broad PAM range and high specificity and activity. Nat Biotechnol2020;
38:1154–8
137.
KleinstiverBPPrewMSTsaiSQTopkarVVNguyenNTZhengZGonzalesAPLiZPetersonRTYehJRAryeeMJJoungJK.Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature2015;
523:481–5
138.
NishimasuHShiXIshiguroSGaoLHiranoSOkazakiSNodaTAbudayyehOOGootenbergJSMoriHOuraSHolmesBTanakaMSekiMHiranoHAburataniHIshitaniRIkawaMYachieNZhangFNurekiO.Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science2018;
361:1259–62
139.
CradickTJFineEJAnticoCJBaoG.CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res2013;
41:9584–92
140.
FuYFodenJAKhayterCMaederMLReyonDJoungJKSanderJD.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol2013;
31:822–6
141.
HsuPDScottDAWeinsteinJARanFAKonermannSAgarwalaVLiYFineEJWuXShalemOCradickTJMarraffiniLABaoGZhangF.DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol2013;
31:827–32
142.
SmithCGoreAYanWAbalde-AtristainLLiZHeCWangYBrodskyRAZhangKChengLYeZ.Whole-Genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell2014;12–5 3
VeresAGosisBSDingQCollinsRRagavendranABrandHErdinSTalkowskiMEMusunuruK.Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell2014;
15:27–30
145.
JinSZongYGaoQZhuZWangYQinPLiangCWangDQiuJLZhangFGaoC.Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science2019;
364:292–5
146.
ZuoESunYWeiWYuanTYingWSunHYuanLSteinmetzLMLiYYangH.Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science2019;
364:289–92
147.
McGrathEShinHZhangLPhueJNWuWWShenRFJangYYRevolloJYeZ.Targeting specificity of APOBEC-based cytosine base editor in human iPSCs determined by whole genome sequencing. Nat Commun2019;
10:5353
148.
JinSFeiHZhuZLuoYLiuJGaoSZhangFChenYHWangYGaoC.Rationally designed APOBEC3B cytosine base editors with improved specificity. Mol Cell2020;
79:728–40.e6
149.
DomanJLRaguramANewbyGALiuDR.Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat Biotechnol2020;
38:620–8
150.
BaileyJThewMBallsM.An analysis of the use of animal models in predicting human toxicology and drug safety. Altern Lab Anim2014;
42:181–99
151.
BaileyJThewMBallsM.Predicting human drug toxicity and safety via animal tests: can any one species predict drug toxicity in any other, and do monkeys help?Altern Lab Anim2015;
43:393–403
152.
Corrigan-CurayJO'ReillyMKohnDBCannonPMBaoGBushmanFDCarrollDCathomenTJoungJKRothDSadelainMScharenbergAMvon KalleCZhangFJambouRRosenthalEHassaniMSinghAPorteusMH.Genome editing technologies: defining a path to clinic. Mol Ther2015;
23:796–806
153.
WuHUchimuraKDonnellyELKiritaYMorrisSAHumphreysBD.Comparative analysis and refinement of human PSC-Derived kidney organoid differentiation with Single-Cell transcriptomics. Cell Stem Cell2018;
23:869–81.e8
154.
WatsonCLMaheMMMuneraJHowellJCSundaramNPolingHMSchweitzerJIVallanceJEMayhewCNSunYGrabowskiGFinkbeinerSRSpenceJRShroyerNFWellsJMHelmrathMA.An in vivo model of human small intestine using pluripotent stem cells. Nat Med2014;
20:1310–4
155.
QuinnPMBuckTMMulderAAOhoninCAlvesCHVosRMBialeckaMvan HerwaardenTvan DijkEHCTalibMFreundCMikkersHMMHoebenRCGoumansMJBoonCJFKosterAJChuva de Sousa LopesSMJostCRWijnholdsJ.Human iPSC-derived retinas recapitulate the fetal CRB1 CRB2 complex formation and demonstrate that photoreceptors and Muller glia are targets of AAV5. Stem Cell Rep2019;
12:906–19
156.
NamSASeoEKimJWKimHWKimHLKimKKimTMJuJHGomezIGUchimuraKHumphreysBDYangCWLeeJYKimJChoDWFreedmanBSKimYK.Graft immaturity and safety concerns in transplanted human kidney organoids. Exp Mol Med2019;
51:1–13
157.
ChengQWeiTFarbiakLJohnsonLTDilliardSASiegwartDJ.Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol2020;
15:313–20
158.
CharlesworthCTDeshpandePSDeverDPCamarenaJLemgartVTCromerMKVakulskasCACollingwoodMAZhangLBodeNMBehlkeMADejeneBCieniewiczBRomanoRLeschBJGomez-OspinaNMantriSPavel-DinuMWeinbergKIPorteusMH.Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med2019;
25:249–54
159.
CakirBXiangYTanakaYKuralMHParentMKangYJChapetonKPattersonBYuanYHeCSRaredonMSBDengelegiJKimKYSunPZhongMLeeSPatraPHyderFNiklasonLELeeSHYoonYSParkIH.Engineering of human brain organoids with a functional vascular-like system. Nat Methods2019;
16:1169–75
160.
TakebeTEnomuraMYoshizawaEKimuraMKoikeHUenoYMatsuzakiTYamazakiTToyoharaTOsafuneKNakauchiHYoshikawaHYTaniguchiH.Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell2015;
16:556–65
161.
SarkerMDNaghiehSSharmaNKChenX.3D biofabrication of vascular networks for tissue regeneration: a report on recent advances. J Pharm Anal2018;
8:277–96
162.
Jiménez-TorresJABeebeDJSungKE.A microfluidic method to mimic luminal structures in the tumor microenvironment. Methods Mol Biol2016;
1458:59–69
163.
Jimenez-TorresJAVirumbrales-MunozMSungKELeeMHAbelEJBeebeDJ.Patient-specific organotypic blood vessels as an in vitro model for anti-angiogenic drug response testing in renal cell carcinoma. EBioMedicine2019;
42:408–19
QianXNguyenHNSongMMHadionoCOgdenSCHammackCYaoBHamerskyGRJacobFZhongCYoonKJJeangWLinLLiYThakorJBergDAZhangCKangEChickeringMNauenDHoCYWenZChristianKMShiPYMaherBJWuHJinPTangHSongHMingGL.Brain-Region-Specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell2016;
165:1238–54
168.
ReidJAPalmerXLMollicaPANorthamNSachsPCBrunoRD.A 3D bioprinter platform for mechanistic analysis of tumoroids and chimeric mammary organoids. Sci Rep2019;
9:7466
169.
Marti-FigueroaCRAshtonRS.The case for applying tissue engineering methodologies to instruct human organoid morphogenesis. Acta Biomater2017;
54:35–44
170.
RegierMCMontanez-SauriSISchwartzMPMurphyWLBeebeDJSungKE.The influence of biomaterials on cytokine production in 3D cultures. Biomacromolecules2017;
18:709–18
