Craniosynostosis is a rare congenital bone condition where skull sutures fuse prematurely and is linked to mutations in over 60 genes. Generating mutation-specific in vitro models allows investigation of craniosynostosis-associated mutations without the need for patient-derived material or transgenic gene expression. Here, we developed a human in vitro disease model with the CRISPR-Cas9 prime editing variant, using an immortalized TERT-immortalized mesenchymal bone marrow-derived stem (MSC-TERT) cell line with osteogenic potential. MSC-TERT cells showed a higher resistance to prime editing, compared with HEK293FT cells. Addition of dnMLH1 and epegRNAs resulted in higher editing efficiencies in HEK293FT cells, but not in MSC-TERT cells. Prime editing efficiency varied between targeted loci and was found to be more efficient in nonadherent cells compared with adherent cells. Prime editing continued over 4 days in an isolated nonadherent HEK293FT culture. Our results present a foundation on the use of prime editing to establish FGFR2 mutation-specific in vitro models and their application in MSC-TERT cells.
Get full access to this article
View all access options for this article.
References
1.
CiureaAV, ToaderC. Genetics of craniosynostosis: Review of the literature. J Med Life2009;2(1):5–17.
2.
CohenMM. Sutural biology and the correlates of craniosynostosis. Am J Med Genet1993;47(5):581–616; doi: 10.1002/ajmg.1320470507
3.
JinSW, SimKB, KimSD. Development and growth of the normal cranial vault : an embryologic review. J Korean Neurosurg Soc2016;59(3):192–196; doi: 10.3340/jkns.2016.59.3.192
4.
Morriss-KayGM, WilkieAOM. Growth of the normal skull vault and its alteration in craniosynostosis: Insights from human genetics and experimental studies. J Anat2005;207(5):637–653; doi: 10.1111/j.1469-7580.2005.00475.x
5.
PosnickJC, RuizRL. The craniofacial dysostosis syndromes: Current surgical thinking and future directions. Cleft Palate-Craniofacial J2000;37(5):433; doi: 10.1597/1545-1569(2000)037<0433:TCDSCS>2.0.CO;2
6.
DurhamEL, HowieRN, CrayJJ. Gene/environment interactions in craniosynostosis: A brief review. Orthod Craniofac Res2017;20(Suppl 1):8–11; doi: 10.1111/ocr.12153
TwiggSRF, WilkieAOM. A genetic-pathophysiological framework for craniosynostosis. Am J Hum Genet2015;97(3):359–377; doi: 10.1016/j.ajhg.2015.07.006
13.
JamesAW, XuY, WangR, et al.Proliferation, osteogenic differentiation, and FGF-2 modulation of posterofrontal/sagittal suture–derived mesenchymal cells in vitro. Plast Reconstr Surg2008;122(1):53–63; doi: 10.1097/PRS.0b013e31817747b5
14.
FarmerDT, MlcochovaH, ZhouY, et al.The developing mouse coronal suture at single-cell resolution. Nat Commun2021;12(1):4797; doi: 10.1038/s41467-021-24917-9
15.
BalekL, GudernovaI, VeselaI, et al.ARQ 087 inhibits FGFR signaling and rescues aberrant cell proliferation and differentiation in experimental models of craniosynostoses and chondrodysplasias caused by activating mutations in FGFR1, FGFR2 and FGFR3. Bone2017;105:57–66; doi: 10.1016/j.bone.2017.08.016
16.
ChenH, MaJ, LiW, et al.A molecular brake in the kinase hinge region regulates the activity of receptor tyrosine kinases. Mol Cell2007;27(5):717–730; doi: 10.1016/j.molcel.2007.06.028
17.
MiraouiH, OudinaK, PetiteH, et al.Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J Biol Chem2009;284(8):4897–4904; doi: 10.1074/jbc.M805432200
18.
AhmedZ, SchüllerAC, SuhlingK, et al.Extracellular point mutations in FGFR2 elicit unexpected changes in intracellular signalling. Biochem J2008;413(1):37–49; doi: 10.1042/BJ20071594
19.
NeilsonKM, FrieselRE. Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome. J Biol Chem1995;270(44):26037–26040; doi: 10.1074/jbc.270.44.26037
20.
LeeKKL, PeskettE, QuinnCM, et al.Overexpression of Fgfr2c causes craniofacial bone hypoplasia and ameliorates craniosynostosis in the Crouzon mouse. Dis Model Mech2018;11(11):dmm035311; doi: 10.1242/dmm.035311
21.
SuzukiH, SudaN, ShigaM, et al.Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts. J Cell Physiol2012;227(9):3267–3277; doi: 10.1002/jcp.24021
22.
PamiesD, LeistM, CoeckeS, et al.Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0). ALTEX - Altern Anim Exp2022;39:30–70; doi: 10.14573/altex.2111011
23.
WeiskirchenS, SchröderSK, BuhlEM, et al.A beginner’s guide to cell culture: Practical advice for preventing needless problems. Cells2023;12(5):682; doi: 10.3390/cells12050682
24.
EllJ, RegnS, BuchbergerA-M, et al.Donor-dependent variances of human adipose-derived stem cells in respect to the in-vitro endothelial cell differentiation capability. Adipocyte2017;6(1):20–32; doi: 10.1080/21623945.2016.1273299
25.
ChansardA, DubrulleN, Poujol de MolliensM, et al.Unveiling interindividual variability of human fibroblast innate immune response using robust cell-based protocols. Front Immunol2020;11:569331; doi: 10.3389/fimmu.2020.569331
26.
SimonsenJL, RosadaC, SerakinciN, et al.Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol2002;20(6):592–596; doi: 10.1038/nbt0602-592
27.
AnzaloneAV, RandolphPB, DavisJR, et al.Search-and-replace genome editing without double-strand breaks or donor DNA. Nature2019;576(7785):149–157; doi: 10.1038/s41586-019-1711-4
28.
AnzaloneAV, GaoXD, PodrackyCJ, et al.Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol2022;40(5):731–740; doi: 10.1038/s41587-021-01133-w
29.
ChenPJ, HussmannJA, YanJ, et al.Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell2021;184(22):5635–5652.e29; doi: 10.1016/j.cell.2021.09.018
30.
NelsonJW, RandolphPB, ShenSP, et al.Engineered pegRNAs improve prime editing efficiency. Nat Biotechnol2022;40(3):402–410; doi: 10.1038/s41587-021-01039-7
31.
ClementK, ReesH, CanverMC, et al.CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat Biotechnol2019;37(3):224–226; doi: 10.1038/s41587-019-0032-3
32.
BrinkmanEK, ChenT, AmendolaM, et al.Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res2014;42(22):e168; doi: 10.1093/nar/gku936
33.
KimHK, YuG, ParkJ, et al.Predicting the efficiency of prime editing guide RNAs in human cells. Nat Biotechnol2021;39(2):198–206; doi: 10.1038/s41587-020-0677-y
34.
YuG, KimHK, ParkJ, et al.Prediction of efficiencies for diverse prime editing systems in multiple cell types. Cell2023;186(10):2256–2272.e23; doi: 10.1016/j.cell.2023.03.034
35.
BarrellWB, Adel Al-LamiH, GoosJAC, et al.Identification of a novel variant of the ciliopathic gene FUZZY associated with craniosynostosis. Eur J Hum Genet2022;30(3):282–290; doi: 10.1038/s41431-021-00988-6
36.
ItohN, OrnitzDM. Evolution of the Fgf and Fgfr gene families. Trends Genet2004;20(11):563–569; doi: 10.1016/j.tig.2004.08.007
37.
ThisseB, ThisseC. Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol2005;287(2):390–402; doi: 10.1016/j.ydbio.2005.09.011
38.
LannerF, RossantJ. The role of FGF/Erk signaling in pluripotent cells. Development2010;137(20):3351–3360; doi: 10.1242/dev.050146
39.
van der EerdenBCJ, van der HeydenJC, van HamburgJP, et al.A human vitamin D receptor mutation causes rickets and impaired Th1/Th17 responses. Bone2014;69:6–11; doi: 10.1016/j.bone.2014.08.005
40.
ShoreEM, XuM, FeldmanGJ, et al.A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva. Nat Genet2006;38(5):525–527; doi: 10.1038/ng1783
41.
ChoiJ, ChenW, SuiterCC, et al.Precise genomic deletions using paired prime editing. Nat Biotechnol2022;40(2):218–226; doi: 10.1038/s41587-021-01025-z
42.
LiuB, DongX, ChengH, et al.A split prime editor with untethered reverse transcriptase and circular RNA template. Nat Biotechnol2022;40(9):1388–1393; doi: 10.1038/s41587-022-01255-9
43.
ScheneIF, JooreIP, OkaR, et al.Prime editing for functional repair in patient-derived disease models. Nat Commun2020;11(1):5352; doi: 10.1038/s41467-020-19136-7
44.
ScheneIF, JooreIP, BaijensJHL, et al.Mutation-specific reporter for optimization and enrichment of prime editing. Nat Commun2022;13(1):1028; doi: 10.1038/s41467-022-28656-3
45.
GeurtsMH, de PoelE, Pleguezuelos-ManzanoC, et al.Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids. Life Sci Alliance2021;4(10):e202000940; doi: 10.26508/lsa.202000940
46.
DaerRM, CuttsJP, BrafmanDA, et al.The impact of chromatin dynamics on Cas9-mediated genome editing in human cells. ACS Synth Biol2017;6(3):428–438; doi: 10.1021/acssynbio.5b00299
47.
Kallimasioti-PaziEM, Thelakkad ChathothK, TaylorGC, et al.Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair. PLoS Biol2018;16(12):e2005595; doi: 10.1371/journal.pbio.2005595
HorlbeckMA, WitkowskyLB, GuglielmiB, et al.Nucleosomes impede Cas9 access to DNA in vivo and in vitro. Elife2016;5:e12677; doi: 10.7554/eLife.12677
50.
KnightSC, XieL, DengW, et al.Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science2015;350(6262):823–826; doi: 10.1126/science.aac6572
51.
JensenKT, FløeL, PetersenTS, et al.Chromatin accessibility and guide sequence secondary structure affect CRISPR-Cas9 gene editing efficiency. FEBS Lett2017;591(13):1892–1901; doi: 10.1002/1873-3468.12707
52.
LiG, ZhangX, WangH, et al.Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors. Int J Biochem Cell Biol2020;125:105790; doi: 10.1016/j.biocel.2020.105790
53.
IsaacRS, JiangF, DoudnaJA, et al.Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. Elife2016;5:e13450; doi: 10.7554/eLife.13450
54.
LiX, ChenW, MartinBK, et al.Chromatin context-dependent regulation and epigenetic manipulation of prime editing. Cell2024;187(10):2411–2427.e25; doi: 10.1016/j.cell.2024.03.020
55.
RobertsonSC, MeyerAN, HartKC, et al.Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain. Proc Natl Acad Sci U S A1998;95(8):4567–4572; doi: 10.1073/pnas.95.8.4567
56.
EswarakumarVP, HorowitzMC, LocklinR, et al.A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc Natl Acad Sci U S A2004;101(34):12555–12560; doi: 10.1073/pnas.0405031101
57.
EswarakumarVP, OzcanF, LewED, et al.Attenuation of signaling pathways stimulated by pathologically activated FGF-receptor 2 mutants prevents craniosynostosis. Proc Natl Acad Sci U S A2006;103(49):18603–18608; doi: 10.1073/pnas.0609157103
58.
PeskettE, KumarS, BairdW, et al.Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme. Biol Open2017;6(2):223–231; doi: 10.1242/bio.022178
59.
XuX, WeinsteinM, LiC, et al.Fibroblast growth factor receptor 2 (FGFR2)-mediated reciprocal regulation loop between FGF8 and FGF10 is essential for limb induction. Development1998;125(4):753–765; doi: 10.1242/dev.125.4.753
60.
HajihosseiniMK, WilsonS, De MoerloozeL, et al.A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes. Proc Natl Acad Sci U S A2001;98(7):3855–3860; doi: 10.1073/pnas.071586898
61.
RayAT, MazotP, BrewerJR, et al.FGF signaling regulates development by processes beyond canonical pathways. Genes Dev2020;34(23–24):1735–1752; doi: 10.1101/gad.342956.120
62.
BylundL, KytöläS, LuiWO, et al.Analysis of the cytogenetic stability of the human embryonal kidney cell line 293 by cytogenetic and STR profiling approaches. Cytogenet Genome Res2004;106(1):28–32; doi: 10.1159/000078556
63.
LinY-C, BooneM, MeurisL, et al.Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations. Nat Commun2014;5(1):4767; doi: 10.1038/ncomms5767
64.
LatkoM, CzyrekA, PorębskaN, et al.Cross-talk between fibroblast growth factor receptors and other cell surface proteins. Cells2019;8(5):455; doi: 10.3390/cells8050455
65.
MieczkowskiK, PopedaM, LesniakD, et al.FGFR2 controls growth, adhesion and migration of nontumorigenic human mammary epithelial cells by regulation of integrin β1 degradation. J Mammary Gland Biol Neoplasia2023;28(1):9; doi: 10.1007/s10911-023-09537-x
FanganielloRD, SertiéAL, ReisEM, et al.Apert p.Ser252Trp mutation in FGFR2 alters osteogenic potential and gene expression of cranial periosteal cells. Mol Med2007;13(7–8):422–442; doi: 10.2119/2007-00027.Fanganiello
68.
KaabecheK, GuenouH, BouvardD, et al.Cbl-mediated ubiquitination of α5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation. J Cell Sci2005;118(Pt 6):1223–1232; doi: 10.1242/jcs.01679
69.
Sanchez-HerasE, HowellFV, WilliamsG, et al.The fibroblast growth factor receptor acid box is essential for interactions with N-cadherin and all of the major isoforms of neural cell adhesion molecule. J Biol Chem2006;281(46):35208–35216; doi: 10.1074/jbc.M608655200
70.
RauchA, HaakonssonAK, MadsenJGS, et al.Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis. Nat Genet2019;51(4):716–727; doi: 10.1038/s41588-019-0359-1
71.
LemonnierJ, HaÿE, DelannoyP, et al.Role of N-cadherin and protein kinase c in osteoblast gene activation induced by the S252W fibroblast growth factor receptor 2 mutation in apert craniosynostosis. J Bone Miner Res2001;16(5):832–845; doi: 10.1359/jbmr.2001.16.5.832
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
