Advertisement

CRISPR/Cas9 facilitates genomic editing for large-scale functional studies in pluripotent stem cell cultures

  • Xiao-Fei Li
  • Yong-Wei Zhou
  • Peng-Fei Cai
  • Wei-Cong Fu
  • Jin-Hua Wang
  • Jin-Yang Chen
  • Qi-Ning YangEmail author
Review

Abstract

Pluripotent stem cell (PSC) cultures form an integral part of biomedical and medical research due to their capacity to rapidly proliferate and differentiate into hundreds of highly specialized cell types. This makes them a highly useful tool in exploring human physiology and disease. Genomic editing of PSC cultures is an essential method of attaining answers to basic physiological functions, developing in vitro models of human disease, and exploring potential therapeutic strategies and the identification of drug targets. Achieving reliable and efficient genomic editing is an important aspect of using large-scale PSC cultures. The CRISPR/Cas9 genomic editing tool has facilitated highly efficient gene knockout, gene correction, or gene modifications through the design and use of single-guide RNAs which are delivered to the target DNA via Cas9. CRISPR/Cas9 modification of PSCs has furthered the understanding of basic physiology and has been utilized to develop in vitro disease models, to test therapeutic strategies, and to facilitate regenerative or tissue repair approaches. In this review, we discuss the benefits of the CRISPR/Cas9 system in large-scale PSC cultures.

Notes

Acknowledgements

This work was supported by the Zhejiang Provincial Science and Technology Projects (no. LGF19H060005 to Q. N. Y.).

Author contributions

All authors have participated equally in drafting and revising this paper.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Acun A, Zorlutuna P (2019) CRISPR/Cas9 edited induced pluripotent stem cell-based vascular tissues to model aging and disease-dependent impairment. Tissue Eng Part A 25:759–772.  https://doi.org/10.1089/ten.tea.2018.0271 CrossRefPubMedGoogle Scholar
  2. Anders C, Niewoehner O, Duerst A, Jinek M (2014) Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513:569–573.  https://doi.org/10.1038/nature13579 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Anderson EM, Haupt A, Schiel JA et al (2015) Systematic analysis of CRISPR-Cas9 mismatch tolerance reveals low levels of off-target activity. J Biotechnol 211:56–65.  https://doi.org/10.1016/j.jbiotec.2015.06.427 CrossRefPubMedGoogle Scholar
  4. Barrangou R (2015a) Diversity of CRISPR-Cas immune systems and molecular machines. Genome Biol 16:247CrossRefGoogle Scholar
  5. Barrangou R (2015b) The roles of CRISPR-Cas systems in adaptive immunity and beyond. Curr Opin Immunol 32:36–41CrossRefGoogle Scholar
  6. Barrangou R, Doudna JA (2016) Applications of CRISPR technologies in research and beyond. Nat Biotechnol 34:933–941.  https://doi.org/10.1038/nbt.3659 CrossRefPubMedGoogle Scholar
  7. Batista PJ, Molinie B, Wang J et al (2014) M6A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15:707–719.  https://doi.org/10.1016/j.stem.2014.09.019 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brazelton VA, Zarecor S, Wright DA et al (2015) A quick guide to CRISPR sgRNA design tools. GM Crops Food 6:266–276.  https://doi.org/10.1080/21645698.2015.1137690 CrossRefPubMedGoogle Scholar
  9. Brouns SJJ, Jore MM, Lundgren M et al (2018) Small CRISPR RNAs guide antiviral defense in prokaryotes. HHS Public Access. 321:960–964.  https://doi.org/10.1126/science.1159689.Small CrossRefGoogle Scholar
  10. Cao J, Wu L, Zhang SM et al (2016) An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting. Nucleic Acids Res.  https://doi.org/10.1093/nar/gkw660 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Carroll D (2011) Genome engineering with zinc-finger nucleases. Genetics 188:773–782.  https://doi.org/10.1534/genetics.111.131433 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Chen KG, Mallon BS, McKay RDG, Robey PG (2014) Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics. Cell Stem Cell 14:13–26CrossRefGoogle Scholar
  13. Chen Y, Cao J, Xiong M et al (2015) Engineering human stem cell lines with inducible gene knockout using CRISPR/Cas9. Cell Stem Cell 17:233–244.  https://doi.org/10.1016/j.stem.2015.06.001 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Christian M, Cermak T, Doyle EL et al (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186:757–761.  https://doi.org/10.1534/genetics.110.120717 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Cornu TI, Thibodeau-Beganny S, Guhl E et al (2008) DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases. Mol Ther 16:352–358.  https://doi.org/10.1038/sj.mt.6300357 CrossRefPubMedGoogle Scholar
  16. Cox DBT, Platt RJ, Zhang F (2015) Therapeutic genome editing: prospects and challenges. Nat Med 21:121–131CrossRefGoogle Scholar
  17. Daley GQ (2012) The promise and perils of stem cell therapeutics. Cell Stem Cell 10:740–749CrossRefGoogle Scholar
  18. Ding Q, Lee YK, Schaefer EAK et al (2013a) A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12:238–251.  https://doi.org/10.1016/j.stem.2012.11.011 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Ding Q, Regan SN, Xia Y et al (2013b) Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs. Cell Stem Cell 12:393–394.  https://doi.org/10.1016/j.stem.2013.03.006 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Doudna JA, Charpentier E (2014) The new frontier of genome engineering with CRISPR-Cas9. Science 346(6213):1258096CrossRefGoogle Scholar
  21. Doyon Y, Vo TD, Mendel MC et al (2010) Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat Methods 8:74CrossRefGoogle Scholar
  22. Engle SJ, Puppala D (2013) Integrating human pluripotent stem cells into drug development. Cell Stem Cell 12:669–677CrossRefGoogle Scholar
  23. Foltz LP, Howden SE, Thomson JA, Clegg DO (2018) Functional assessment of patient-derived retinal pigment epithelial cells edited by CRISPR/Cas9. Int J Mol Sci.  https://doi.org/10.3390/ijms19124127 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Freedman BS, Brooks CR, Lam AQ et al (2015) Modelling kidney disease with CRISPR-mutant kidney organoids derived from human pluripotent epiblast spheroids. Nat Commun.  https://doi.org/10.1038/ncomms9715 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Garneau JE, Dupuis M-È, Villion M et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71.  https://doi.org/10.1038/nature09523 CrossRefGoogle Scholar
  26. Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci 109:E2579–E2586.  https://doi.org/10.1073/pnas.1208507109 CrossRefPubMedGoogle Scholar
  27. Gutierrez-Guerrero A, Sanchez-Hernandez S, Galvani G et al (2018) Comparison of zinc finger nucleases versus CRISPR-specific nucleases for genome editing of the Wiskott-Aldrich syndrome locus. Hum Gene Ther 29:366–380.  https://doi.org/10.1089/hum.2017.047 CrossRefPubMedGoogle Scholar
  28. Hendel A, Bak RO, Clark JT et al (2015) Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat Biotechnol 33:985–989.  https://doi.org/10.1038/nbt.3290 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Hockemeyer D, Wang H, Kiani S et al (2011) Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol 29:731–734.  https://doi.org/10.1038/nbt.1927 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Hryhorowicz M, Lipinski D, Zeyland J, Slomski R (2017) CRISPR/Cas9 immune system as a tool for genome engineering. Arch Immunol Ther Exp (Warsz) 65:233–240.  https://doi.org/10.1007/s00005-016-0427-5 CrossRefGoogle Scholar
  31. Huang N, Huang Z, Gao M et al (2018) Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases. J Exp Clin Cancer Res 37:62.  https://doi.org/10.1186/s13046-018-0732-4 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Jacob A, Morley M, Hawkins F et al (2017) Differentiation of human pluripotent stem cells into functional lung alveolar epithelial cells. Cell Stem Cell 21:472–488.  https://doi.org/10.1016/j.stem.2017.08.014 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821CrossRefGoogle Scholar
  34. Jinek M, East A, Cheng A et al (2013) RNA-programmed genome editing in human cells. Elife.  https://doi.org/10.7554/elife.00471 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Joung JK, Sander JD (2013) TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol 14:49–55.  https://doi.org/10.1038/nrm3486.talens CrossRefPubMedGoogle Scholar
  36. Kim Y-G, Chandrasegaran S (1994) Chimeric restriction endonuclease (Flavobacterium okeanokoites/Escherichia cofi/hybrid restriction endonuclease/protein engineering/recognition and cleavage domains). Biochemistry 91:883–887Google Scholar
  37. Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci USA 93:1156–1160CrossRefGoogle Scholar
  38. Lannagan TRM, Lee YK, Wang T et al (2019) Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis. Gut 68:684–692.  https://doi.org/10.1136/gutjnl-2017-315920 CrossRefPubMedGoogle Scholar
  39. Lee J, Bayarsaikhan D, Arivazhagan R et al (2019) CRISPR/Cas9 edited sRAGE-MSCs protect neuronal death in Parkinson’s disease model. Int J Stem Cells 12:114–124.  https://doi.org/10.15283/ijsc18110 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Li L, Wv LP, Chandrasegaran S (1992) Functional domains in Fok I restriction endonuclease. Biochemistry 89:4275–4279Google Scholar
  41. Liu Y, Deng W (2016) Reverse engineering human neurodegenerative disease using pluripotent stem cell technology. Brain Res 1638:30–41CrossRefGoogle Scholar
  42. Liu B, Saber A, Haisma HJ (2019) CRISPR/Cas9: a powerful tool for identification of new targets for cancer treatment. Drug Discov Today 24:955–970.  https://doi.org/10.1016/j.drudis.2019.02.011 CrossRefPubMedGoogle Scholar
  43. Maeder ML, Thibodeau-Beganny S, Osiak A et al (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31:294–301.  https://doi.org/10.1016/j.molcel.2008.06.016 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Makarova KS, Grishin NV, Shabalina SA et al (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7.  https://doi.org/10.1186/1745-6150-1-7 CrossRefPubMedPubMedCentralGoogle Scholar
  45. Makarova KS, Haft DH, Barrangou R et al (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9:467–477.  https://doi.org/10.1038/nrmicro2577 CrossRefPubMedGoogle Scholar
  46. Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science (80-) 339:823–826.  https://doi.org/10.1126/science.1232033 CrossRefGoogle Scholar
  47. Mandal PK, Ferreira LMR, Collins R et al (2014) Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15:643–652.  https://doi.org/10.1016/j.stem.2014.10.004 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Mandegar MA, Huebsch N, Frolov EB et al (2016) CRISPR interference efficiently induces specific and reversible gene silencing in human iPSCs. Cell Stem Cell 18:541–553.  https://doi.org/10.1016/j.stem.2016.01.022 CrossRefPubMedPubMedCentralGoogle Scholar
  49. McKee C, Chaudhry GR (2017) Advances and challenges in stem cell culture. Colloids Surfaces B Biointerfaces 159:62–77CrossRefGoogle Scholar
  50. Merkert S, Martin U (2016) Targeted genome engineering using designer nucleases: state of the art and practical guidance for application in human pluripotent stem cells. Stem Cell Res 16:377–386.  https://doi.org/10.1016/J.SCR.2016.02.027 CrossRefPubMedGoogle Scholar
  51. Miller JC, Holmes MC, Wang J et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785.  https://doi.org/10.1038/nbt1319 CrossRefPubMedGoogle Scholar
  52. Mohamed N-V, Larroquette F, Beitel LK et al (2019) One step into the future: new iPSC tools to advance research in parkinson’s disease and neurological disorders. J Parkinsons Dis.  https://doi.org/10.3233/jpd-181515 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Mohr SE, Hu Y, Ewen-Campen B et al (2016) CRISPR guide RNA design for research applications. FEBS J 283:3232–3238CrossRefGoogle Scholar
  54. Mojica FJM, Montoliu L (2016) On the origin of CRISPR-Cas technology: from prokaryotes to mammals. Trends Microbiol 24:811–820CrossRefGoogle Scholar
  55. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740.  https://doi.org/10.1099/mic.0.023960-0 CrossRefPubMedGoogle Scholar
  56. Morizane A, Doi D, Kikuchi T et al (2013) Direct comparison of autologous and allogeneic transplantation of IPSC-derived neural cells in the brain of a nonhuman primate. Stem Cell Rep 1:283–292.  https://doi.org/10.1016/j.stemcr.2013.08.007 CrossRefGoogle Scholar
  57. Motta BM, Pramstaller PP, Hicks AA, Rossini A (2017) The impact of CRISPR/Cas9 technology on cardiac research: from disease modelling to therapeutic approaches. Stem Cells Int 2017:1–13.  https://doi.org/10.1155/2017/8960236 CrossRefGoogle Scholar
  58. Nakano C, Kitabatake Y, Takeyari S et al (2019) Genetic correction of induced pluripotent stem cells mediated by transcription activator-like effector nucleases targeting ALPL recovers enzyme activity and calcification in vitro. Mol Genet Metab 127:158–165.  https://doi.org/10.1016/j.ymgme.2019.05.014 CrossRefPubMedGoogle Scholar
  59. Nii T, Kohara H, Marumoto T et al (2016) Single-cell-state culture of human pluripotent stem cells increases transfection efficiency. Biores Open Access 5:127–136.  https://doi.org/10.1089/biores.2016.0009 CrossRefPubMedPubMedCentralGoogle Scholar
  60. Ormond KE, Mortlock DP, Scholes DT et al (2017) Human germline genome editing. Am J Hum Genet 101:167–176.  https://doi.org/10.1016/j.ajhg.2017.06.012 CrossRefPubMedPubMedCentralGoogle Scholar
  61. Ramirez CL, Foley JE, Wright DA et al (2008) Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5:374CrossRefGoogle Scholar
  62. Ran F, Hsu P, Wright J et al (2013) Genome engineering using crispr-cas9 system. Nature protocols. Nature Publishing Group, London, pp 2281–2308Google Scholar
  63. Rath D, Amlinger L, Rath A, Lundgren M (2015) The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie 117:119–128.  https://doi.org/10.1016/J.BIOCHI.2015.03.025 CrossRefPubMedGoogle Scholar
  64. Reubinoff BE, Pera MF, Fong C-Y et al (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404.  https://doi.org/10.1038/74447 CrossRefPubMedGoogle Scholar
  65. Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JDJJ (2013) FLASH assembly of TALENs enables high-throughput genome editing. Nat Biotechnol 30:460–465.  https://doi.org/10.1038/nbt.2170.FLASH CrossRefGoogle Scholar
  66. Schmidt F, Grimm D (2015) CRISPR genome engineering and viral gene delivery: a case of mutual attraction. Biotechnol J 10:258–272CrossRefGoogle Scholar
  67. Schuster S, Saravanakumar S, Schöls L, Hauser S (2019) Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for the STUB1 locus. Stem Cell Res.  https://doi.org/10.1016/j.scr.2018.101378 CrossRefPubMedGoogle Scholar
  68. Shalem O, Sanjana NE, Hartenian E et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science (80-) 343:84–87.  https://doi.org/10.1126/science.1247005 CrossRefGoogle Scholar
  69. Smith J (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28:3361–3369.  https://doi.org/10.1093/nar/28.17.3361 CrossRefPubMedPubMedCentralGoogle Scholar
  70. Sohn EH, Jiao C, Kaalberg E et al (2015) Allogenic iPSC-derived RPE cell transplants induce immune response in pigs: a pilot study. Sci Rep 5:11791CrossRefGoogle Scholar
  71. Sterneckert JL, Reinhardt P, Schöler HR (2014) Investigating human disease using stem cell models. Nat Rev Genet 15:625–639CrossRefGoogle Scholar
  72. Sun S, Xiao J, Huo J et al (2018) Targeting ectodysplasin promotor by CRISPR/dCas9-effector effectively induces the reprogramming of human bone marrow-derived mesenchymal stem cells into sweat gland-like cells. Stem Cell Res Ther 9:1–10.  https://doi.org/10.1186/s13287-017-0758-0 CrossRefGoogle Scholar
  73. Sun J, Carlson-Stevermer J, Das U et al (2019) CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage. Nat Commun.  https://doi.org/10.1038/s41467-018-07971-8 CrossRefPubMedPubMedCentralGoogle Scholar
  74. Suzuki S, Sargent RG, Illek B et al (2016) TALENs facilitate single-step seamless SDF correction of F508del CFTR in airway epithelial submucosal gland cell-derived CF-iPSCs. Mol Ther Nucleic Acids 5:e273.  https://doi.org/10.1038/mtna.2015.43 CrossRefPubMedPubMedCentralGoogle Scholar
  75. Tadić V, Josipović G, Zoldoš V, Vojta A (2019) CRISPR/Cas9-based epigenome editing: an overview of dCas9-based tools with special emphasis on off-target activity. Methods.  https://doi.org/10.1016/j.ymeth.2019.05.003 CrossRefPubMedGoogle Scholar
  76. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676.  https://doi.org/10.1016/j.cell.2006.07.024 CrossRefGoogle Scholar
  77. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147.  https://doi.org/10.1126/science.282.5391.1145 CrossRefGoogle Scholar
  78. Trounson A, McDonald C (2015) Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell 17:11–22.  https://doi.org/10.1016/j.stem.2015.06.007 CrossRefPubMedGoogle Scholar
  79. Urnov FD, Rebar EJ, Holmes MC et al (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646CrossRefGoogle Scholar
  80. Vandamme TF (2014) Use of rodents as models of human diseases. J Pharm Bioallied Sci 6:2–9.  https://doi.org/10.4103/0975-7406.124301 CrossRefPubMedPubMedCentralGoogle Scholar
  81. Vercoe RB, Chang JT, Dy RL et al (2013) Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands. PLoS Genet.  https://doi.org/10.1371/journal.pgen.1003454 CrossRefPubMedPubMedCentralGoogle Scholar
  82. Wang Q, Zou Y, Nowotschin S et al (2017) The p53 family coordinates Wnt and nodal inputs in mesendodermal differentiation of embryonic stem cells. Cell Stem Cell 20:70–86.  https://doi.org/10.1016/j.stem.2016.10.002 CrossRefPubMedGoogle Scholar
  83. Wang S, Min Z, Ji Q et al (2019) Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction. Protein Cell.  https://doi.org/10.1007/s13238-019-0623-2 CrossRefPubMedPubMedCentralGoogle Scholar
  84. Wei H, Zhang XH, Clift C et al (2018) CRISPR/Cas9 Gene editing of RyR2 in human stem cell-derived cardiomyocytes provides a novel approach in investigating dysfunctional Ca 2 + signaling. Cell Calcium 73:104–111.  https://doi.org/10.1016/j.ceca.2018.04.009 CrossRefPubMedPubMedCentralGoogle Scholar
  85. Wong CH, Siah KW, Lo AW (2019) Estimation of clinical trial success rates and related parameters. Biostatistics 20:273–286.  https://doi.org/10.1093/biostatistics/kxx069 CrossRefPubMedGoogle Scholar
  86. Zhang F, Wen Y, Guo X (2014) CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet.  https://doi.org/10.1093/hmg/ddu125 CrossRefPubMedPubMedCentralGoogle Scholar
  87. Zhang Z, Zhang Y, Gao F et al (2017) CRISPR/Cas9 genome-editing system in human stem cells: current status and future prospects. Mol Ther Nucleic Acids 9:230–241CrossRefGoogle Scholar
  88. Zhu W, Zhang B, Li M et al (2019) Precisely controlling endogenous protein dosage in hPSCs and derivatives to model FOXG1 syndrome. Nat Commun.  https://doi.org/10.1038/s41467-019-08841-7 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Joint SurgeryJinhua Municipal Central HospitalJinhuaPeople’s Republic of China
  2. 2.Research and Development DepartmentZhejiang Healthfuture Institute for Cell-Based Applied TechnologyHangzhouPeople’s Republic of China

Personalised recommendations