Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼33% correctly targeted clones) compared to conventional targeting protocol (∼3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.
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References
1.
TakahashiK and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
2.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
3.
YuJ, VodyanikMA, Smuga-OttoK, Antosiewicz-BourgetJ, FraneJL, TianS, NieJ, JonsdottirGA, RuottiV, et al. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318:1917–1920.
4.
ZwakaTP and ThomsonJA. (2003). Homologous recombination in human embryonic stem cells. Nat Biotechnol, 21:319–321.
5.
DavisRP, NgES, CostaM, MossmanAK, SourrisK, ElefantyAG and StanleyEG. (2008). Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak-like cells and enables isolation of primitive hematopoietic precursors. Blood, 111:1876–1884.
6.
RubyKM and ZhengB. (2009). Gene targeting in a HUES line of human embryonic stem cells via electroporation. Stem Cells, 27:1496–1506.
7.
XueH, WuS, PapadeasST, SpustaS, SwistowskaAM, MacArthurCC, MattsonMP, MaragakisNJ, CapecchiMR, et al. (2009). A targeted neuroglial reporter line generated by homologous recombination in human embryonic stem cells. Stem Cells, 27:1836–1846.
8.
LiuY, JiangP and DengW. (2011). OLIG gene targeting in human pluripotent stem cells for motor neuron and oligodendrocyte differentiation. Nat Protoc, 6:640–655.
9.
HockemeyerD, SoldnerF, BeardC, GaoQ, MitalipovaM, DeKelverRC, KatibahGE, AmoraR, BoydstonEA, et al. (2009). Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol, 27:851–857.
10.
SchinzelRT, AhfeldtT, LauFH, LeeYK, CowleyA, ShenT, PetersD, LumDH and CowanCA. (2011). Efficient culturing and genetic manipulation of human pluripotent stem cells. PLoS One, 6:e27495.
11.
WangP, RodriguezRT, WangJ, GhodasaraA and KimSK. (2011). Targeting SOX17 in human embryonic stem cells creates unique strategies for isolating and analyzing developing endoderm. Cell Stem Cell, 8:335–346.
12.
GoulburnAL, AldenD, DavisRP, MicallefSJ, NgES, YuQC, LimSM, SohCL, ElliottDA, et al. (2011). A targeted NKX2.1 human embryonic stem cell reporter line enables identification of human basal forebrain derivatives. Stem Cells, 29:462–473.
13.
AizawaE, HirabayashiY, IwanagaY, SuzukiK, SakuraiK, ShimojiM, AibaK, WadaT, TooiN, et al. (2012). Efficient and accurate homologous recombination in hESCs and hiPSCs using helper-dependent adenoviral vectors. Mol Ther, 20:424–431.
14.
BrafmanDA, MoyaN, Allen-SolteroS, FellnerT, RobinsonM, McMillenZL, GaasterlandT and WillertK. (2013). Analysis of SOX2-expressing cell populations derived from human pluripotent stem cells. Stem Cell Reports, 1:464–478.
15.
JinekM, ChylinskiK, FonfaraI, HauerM, DoudnaJA and CharpentierE. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337:816–821.
16.
WangH, YangH, ShivalilaCS, DawlatyMM, ChengAW, ZhangF and JaenischR. (2013). One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 153:910–918.
17.
MaliP, YangL, EsveltKM, AachJ, GuellM, DiCarloJE, NorvilleJE and ChurchGM. (2013). RNA-guided human genome engineering via Cas9. Science, 339:823–826.
18.
HwangWY, FuY, ReyonD, MaederML, TsaiSQ, SanderJD, PetersonRT, YehJR and JoungJK. (2013). Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 31:227–229.
19.
FriedlandAE, TzurYB, EsveltKM, ColaiacovoMP, ChurchGM and CalarcoJA. (2013). Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 10:741–743.
20.
DiCarloJE, NorvilleJE, MaliP, RiosX, AachJ and ChurchGM. (2013). Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res, 41:4336–4343.
NiuY, ShenB, CuiY, ChenY, WangJ, WangL, KangY, ZhaoX, SiW, et al. (2014). Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell, 156:836–843.
23.
ShanerNC, CampbellRE, SteinbachPA, GiepmansBN, PalmerAE and TsienRY. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol, 22:1567–1572.
24.
YuC, LiuY, MaT, LiuK, XuS, ZhangY, LiuH, La RussaM, XieM, DingS and QiLS. (2015). Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell, 16:142–147.
25.
ChuVT, WeberT, WefersB, WurstW, SanderS, RajewskyK and KuhnR. (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol, 33:543–548.
26.
XueH, WuJ, LiS, RaoMS and LiuY. (2016). Genetic modification in human pluripotent stem cells by homologous recombination and CRISPR/Cas9 system. Methods Mol Biol, 1307:173–190.
27.
MacarthurCC, XueH, Van HoofD, LieuPT, DudasM, FontesA, SwistowskiA, TouboulT, SeerkeR, et al. (2012). Chromatin insulator elements block transgene silencing in engineered human embryonic stem cell lines at a defined chromosome 13 locus. Stem Cells Dev, 21:191–205.
28.
SanderJD, ZabackP, JoungJK, VoytasDF and DobbsD. (2007). Zinc finger targeter (ZiFiT): an engineered zinc finger/target site design tool. Nucleic Acids Res, 35:W599–W605.
LiS, XueH, LongB, SunL, TruongT and LiuY. (2015). Efficient generation of hiPSC neural lineage specific knockin reporters using the CRISPR/Cas9 and Cas9 double nickase system. J Vis Exp. e52539; DOI: 10.3791/52539.
31.
WeberE, GruetznerR, WernerS, EnglerC and MarillonnetS. (2011). Assembly of designer TAL effectors by Golden Gate cloning. PLoS One, 6:e19722.
32.
WeberE, EnglerC, GruetznerR, WernerS and MarillonnetS. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS One, 6:e16765.
33.
EnglerC, GruetznerR, KandziaR and MarillonnetS. (2009). Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One, 4:e5553.
34.
GuschinDY, WaiteAJ, KatibahGE, MillerJC, HolmesMC and RebarEJ. (2010). A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649:247–256.
35.
RanFA, HsuPD, WrightJ, AgarwalaV, ScottDA and ZhangF. (2013). Genome engineering using the CRISPR-Cas9 system. Nat Protoc, 8:2281–2308.
36.
XiaoA, ChengZ, KongL, ZhuZ, LinS, GaoG and ZhangB. (2014). CasOT: a genome-wide Cas9/gRNA off-target searching tool. Bioinformatics, 30:1180–1182.
37.
ReinhardtP, GlatzaM, HemmerK, TsytsyuraY, ThielCS, HoingS, MoritzS, PargaJA, WagnerL, et al. (2013). Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling. PLoS One, 8:e59252.
38.
SwistowskiA, PengJ, HanY, SwistowskaAM, RaoMS and ZengX. (2009). Xeno-free defined conditions for culture of human embryonic stem cells, neural stem cells and dopaminergic neurons derived from them. PLoS One, 4:e6233.
FuY, SanderJD, ReyonD, CascioVM and JoungJK. (2014). Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol, 32:279–284.
41.
LiuY, WuY, LeeJC, XueH, PevnyLH, KaprielianZ and RaoMS. (2002). Oligodendrocyte and astrocyte development in rodents: an in situ and immunohistological analysis during embryonic development. Glia, 40:25–43.
42.
MaYC, SongMR, ParkJP, Henry HoHY, HuL, KurtevMV, ZiegJ, MaQ, PfaffSL and GreenbergME. (2008). Regulation of motor neuron specification by phosphorylation of neurogenin 2. Neuron, 58:65–77.
43.
FuY, ReyonD and JoungJK. (2014). Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs. Methods Enzymol, 546:21–45.
44.
CapecchiMR. (2005). Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet, 6:507–512.
45.
FolgerKR, WongEA, WahlG and CapecchiMR. (1982). Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules. Mol Cell Biol, 2:1372–1387.
46.
BaeS, ParkJ and KimJS. (2014). Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics, 30:1473–1475.
47.
KimD, BaeS, ParkJ, KimE, KimS, YuHR, HwangJ, KimJI and KimJS. (2015). Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods, 12:237–243.
48.
WangX, WangY, WuX, WangJ, WangY, QiuZ, ChangT, HuangH, LinRJ and YeeJK. (2015). Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol, 33:175–178.
49.
RanFA, HsuPD, LinCY, GootenbergJS, KonermannS, TrevinoAE, ScottDA, InoueA, MatobaS, ZhangY and ZhangF. (2013). Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell, 154:1380–1389.
50.
MaliP, AachJ, StrangesPB, EsveltKM, MoosburnerM, KosuriS, YangL and ChurchGM. (2013). CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol, 31:833–838.
51.
TsaiSQ, WyvekensN, KhayterC, FodenJA, ThaparV, ReyonD, GoodwinMJ, AryeeMJ and JoungJK. (2014). Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol, 32:569–576.
52.
GuilingerJP, ThompsonDB and LiuDR. (2014). Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol, 32:577–582.
53.
SeilerC, DavuluriG, AbramsJ, ByfieldFJ, JanmeyPA and PackM. (2012). Smooth muscle tension induces invasive remodeling of the zebrafish intestine. PLoS Biol, 10:e1001386.
54.
BairdGS, ZachariasDA and TsienRY. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A, 97:11984–11989.
55.
GilbertLA, HorlbeckMA, AdamsonB, VillaltaJE, ChenY, WhiteheadEH, GuimaraesC, PanningB, PloeghHL, et al. (2014). Genome-scale CRISPR-mediated control of gene repression and activation. Cell, 159:647–661.
56.
KonermannS, BrighamMD, TrevinoAE, JoungJ, AbudayyehOO, BarcenaC, HsuPD, HabibN, GootenbergJS, et al. (2015). Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature, 517:583–588.
MaQ, KintnerC and AndersonDJ. (1996). Identification of neurogenin, a vertebrate neuronal determination gene. Cell, 87:43–52.
59.
FodeC, GradwohlG, MorinX, DierichA, LeMeurM, GoridisC and GuillemotF. (1998). The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron, 20:483–494.
60.
FodeC, MaQ, CasarosaS, AngSL, AndersonDJ and GuillemotF. (2000). A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons. Genes Dev, 14:67–80.
61.
MizuguchiR, SugimoriM, TakebayashiH, KosakoH, NagaoM, YoshidaS, NabeshimaY, ShimamuraK and NakafukuM. (2001). Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron, 31:757–771.
62.
NovitchBG, ChenAI and JessellTM. (2001). Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron, 31:773–789.
63.
ScardigliR, SchuurmansC, GradwohlG and GuillemotF. (2001). Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord. Neuron, 31:203–217.
64.
DixitR, WilkinsonG, CancinoGI, ShakerT, AdnaniL, LiS, DennisD, KurraschD, ChanJA, et al. (2014). Neurog1 and neurog2 control two waves of neuronal differentiation in the piriform cortex. J Neurosci, 34:539–553.
65.
RoybonL, DeierborgT, BrundinP and LiJY. (2009). Involvement of Ngn2, Tbr and NeuroD proteins during postnatal olfactory bulb neurogenesis. Eur J Neurosci, 29:232–243.
66.
RoybonL, HjaltT, StottS, GuillemotF, LiJY and BrundinP. (2009). Neurogenin2 directs granule neuroblast production and amplification while NeuroD1 specifies neuronal fate during hippocampal neurogenesis. PLoS One, 4:e4779.
67.
MaQ, ChenZ, del Barco BarrantesI, de la PompaJL and AndersonDJ. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron, 20:469–482.
68.
ZirlingerM, LoL, McMahonJ, McMahonAP and AndersonDJ. (2002). Transient expression of the bHLH factor neurogenin-2 marks a subpopulation of neural crest cells biased for a sensory but not a neuronal fate. Proc Natl Acad Sci U S A, 99:8084–8089.
69.
HindleyC, AliF, McDowellG, ChengK, JonesA, GuillemotF and PhilpottA. (2012). Post-translational modification of Ngn2 differentially affects transcription of distinct targets to regulate the balance between progenitor maintenance and differentiation. Development, 139:1718–1723.
70.
LeeSK, LeeB, RuizEC and PfaffSL. (2005). Olig2 and Ngn2 function in opposition to modulate gene expression in motor neuron progenitor cells. Genes Dev, 19:282–294.
71.
ShimizuT, NakazawaM, KaniS, BaeYK, ShimizuT, KageyamaR and HibiM. (2010). Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain. Development, 137:1875–1885.
72.
BusskampV, LewisNE, GuyeP, NgAH, ShipmanSL, ByrneSM, SanjanaNE, MurnJ, LiY, et al. (2014). Rapid neurogenesis through transcriptional activation in human stem cells. Mol Syst Biol, 10:760.
73.
BerningerB, CostaMR, KochU, SchroederT, SutorB, GrotheB and GotzM. (2007). Functional properties of neurons derived from in vitro reprogrammed postnatal astroglia. J Neurosci, 27:8654–8664.
74.
MazzoniEO, MahonyS, ClosserM, MorrisonCA, NedelecS, WilliamsDJ, AnD, GiffordDK and WichterleH. (2013). Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity. Nat Neurosci, 16:1219–1227.
75.
LiuML, ZangT, ZouY, ChangJC, GibsonJR, HuberKM and ZhangCL. (2013). Small molecules enable neurogenin 2 to efficiently convert human fibroblasts into cholinergic neurons. Nat Commun, 4:2183.
76.
ZhangY, PakC, HanY, AhleniusH, ZhangZ, ChandaS, MarroS, PatzkeC, AcunaC, et al. (2013). Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron, 78:785–798.
77.
PerrinFE, BonifaceG, SergueraC, LonjonN, SerreA, PrietoM, MalletJ and PrivatA. (2010). Grafted human embryonic progenitors expressing neurogenin-2 stimulate axonal sprouting and improve motor recovery after severe spinal cord injury. PLoS One, 5:e15914.
78.
KrentzNA, NianC and LynnFC. (2014). TALEN/CRISPR-mediated eGFP knock-in add-on at the OCT4 locus does not impact differentiation of human embryonic stem cells towards endoderm. PLoS One, 9:e114275.
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